General description of mitochondrial pathology. Clinical presentation, diagnostics, treatment.

 

General characteristic of multifactorial diseases. Definition of hereditary predisposition. Prevention measures. Levels and ways of prevention of hereditary diseases.

 

Medical genetic counseling. Methods of prenatal diagnostics. Screening programmes.

 

Mitochondria: Energy Conversion

A mitochondrion is a semiautonomous, self-reproducing organelle in the cytoplasm of eukaryotic cells. It has a diameter of 1–2 μm and contains multiple copies of circular mitochondrial DNA (mtDNA) of 16569 base pairs in man. The number of mitochondria per cell and their shape differ in different cell types and can change. An average eukaryotic cell contains 103–104 copies of mitchondria. Mitochondria in animal cells and chloroplasts in plant cells are the sites of essential energy-delivering processes, chloroplasts also being the sites of photosynthesis. HumanmtDNAencodes 13 proteins of the respiratory chain.

 

A. Principal events in mitochondria

Each mitochondrion is surrounded by two highly specialized membranes, the outer and inner membranes. The inner membrane is folded into numerous cristae and encloses the matrix space. The essential energy-generating process in mitochondria is oxidative phosphorylation (OXPHOS). Relatively simple energy carriers such as NADH and FADH2 (nicotinamide adenine dinucleotide in the reduced form and flavin adenine dinucleotide in the reduced form) are produced from the degradation of carbohydrates, fats, and other foodstuffs by oxidation. The important energy carrier adenosine triphosphate (ATP) is formed by oxidative phosphorylation of adenosine diphosphate (ADP) through a series of biochemical reactions in the inner membrane of mitochondria (respiratory chain). Another important function is intracellular oxygen transfer.

B. Oxidative phosphorylation in mitochondria

Adenosine triphosphate (ATP) plays a central role in the conversion of energy in biological systems. It is formed from NADH (nicotinamide adenine dinucleotide) and adenosine diphosphate (ADP) by oxidative phosphorylation (OXPHOS). ATP is a nucleotide consisting of adenine, a ribose, and a triphosphate unit. It is energy-rich because the triphosphate unit contains two phospho-anhydride bonds. Energy (free energy) is released when ATP is hydrolyzed to form ADP. The energy contained in ATP and bound to phosphate is released, for example, during muscle contraction.

C. Electron transfer in the inner mitochondrial membrane

The genomes of mitochondria and chloroplasts contain genes for the formation of the different components of the respiratory chain and oxidative phosphorylation. Three enzyme complexes regulate electron transfer: the NADH-dehydrogenase complex, the b–c1 complex, and the cytochrome oxidase complex (C). Intermediaries are quinone (Q) derivatives such as ubiquinone and cytochrome c. Electron transport leads to the formation of protons (H+). These lead to the conversion of ADP and Pi (inorganic phosphate) into ATP (oxidative phosphorylation). ATP represents a phosphate-bound reservoir of energy, which serves as an energy supplier for all biological systems. This is the reason why genetic defects in mitochondria become manifest primarily as diseases with reduced muscle strength and other degenerative signs.

 

Chloroplasts and Mitochondria

 

Chloroplasts are organelles of plant cells. In contrast to mitochondria, chloroplasts contain a third membrane, the thylakoid membrane. On this membrane, photosynthesis takes place. Chloroplasts and mitochondria of eukaryotic cells contain genomes of circular DNA. Chloroplast DNA ranges fromabout 120000 to 160000 base pairs, depending on the species. It encodes about 120 genes, of which half are involved in DNA-processing functions (transcription, translation, rRNAs, tRNAs, RNA polymerase subunits, and ribosomal proteins). The base sequences of several chloroplast DNAs have been determined. About 12000 base pairs (12 kb) of the genomes of chloroplasts and mitochondria are homologous. Chloroplasts are assumed to be descendants of endosymbiotic cyanobacteria.

 

A. Genes in the chloroplasts of a moss

Genes in the chloroplast genomes are interrupted and contain introns. Each chloroplast contains about 20–40 copies of chloroplast DNA (ctDNA), and there about are 20–40 chloroplasts per cell. The M. polymorphia chloroplast genome contains about 120 genes. Among these are genes for two copies each of four ribosomal RNAs (16 S rRNA, 23 S rRNA, 4.5 S rRNA, and 5 S rRNA). The genes for ribosomal RNA are located in two DNA segments with opposite orientation (inverted repeats), which are characteristic of chloroplast genomes. An 18–19-kb segment with short single-gene copies lies between the two inverted repeats. The genomes of chloroplasts contain genetic information for about 30 tRNAs and about 50 proteins. The proteins belong to photosystem I (two genes), photosystem II (seven genes), the cytochrome system (three genes), and the H+- ATPase system (six genes). The NADH dehydrogenase complex is coded for by six genes; ferredoxin by three genes, and ribulose by one gene. Many of the ribosomal proteins are homologous to those of E. coli. (Figure adapted from Alberts et al, 1994.)

 

Differences between the universal genetic code and mitochondrial codes

Codon	Universal code	Mitochondrial codes
		Mammals	Invertebrates	Yeasts	Plants
UGA	Stop	Trp	Trp	Trp	Stop
AUA	Ile	Met	Met	Met	Ile
CUA	Leu	Leu	Leu	Thr	Leu
AGA/AGG	Arg	Stop	Ser	Arg	Arg

(Data from Alberts et al, 2002)

 

The mitochondrial genome of yeast is large (120 kb). Its genes contain introns. It contains genes for the tRNAs, for the respiratory chain (cytochrome oxidase 1, 2, and 3; cytochrome b), for 15 S and 21 S rRNA, and for subunits 6, 8, and 9 of the ATPase system. The yeast mitochondrial genome is remarkable because its ribosomal RNA genes are separated. The gene for 21 S rRNA contains an intron. About 25% of the mitochondrial genome of yeast contains AT-rich DNA without a coding function.

The genetic code of the mitochondrial genome differs from the universal code iuclear DNA with respect to usage of some codons. The nuclear stop codon UGA codes for tryptophan in mitochondria, while the nuclear codons for arginine (AGA and AGG) function as stop codons in mammalian mitochondria.

 

The Mitochondrial Genome of Man

 

The mitochondrial genome in mammals is small and compact. It contains no introns, and in some regions the genes overlap, so that practically every base pair is part of a coding gene. The mitochondrial genomes of humans and mice have been sequenced and contain extensive homologies. Each consists of about 16.5 kb, i.e., they are considerably smaller than a yeast mitochondrial or a chloroplast genome. In germ cells, mitochondria are almost exclusively present in oocytes, whereas spermatozoa contain few. Thus, they are inherited from the mother, through an oocyte (maternal inheritance).

 

A. Mitochondrial genes in man

 

The human mitochondrial genome, sequenced in 1981 by Andersen et al., has 16569 base pairs.Each mitochondrion contains 2–10 DNA molecules. A heavy (H) and a light (L) single strand can be differentiated by a density gradient. Human mtDNA contains 13 proteincoding regions for four metabolic processes: (i) for NADH dehydrogenase; (ii) for the cytochrome c oxidase complex (subunits 1, 2, and 3); (iii) for cytochrome b; and (iv) for subunits 6 and 8 of the ATPase complex. Unlike that of yeast, mammalian mitochondrial DNA contains seven subunits for NADH dehydrogenase (ND1, ND2, ND3, ND4L, ND4, ND5, and ND6). Of the mitochondrial coding capacity, 60% is taken up by the seven subunits of NADH reductase (ND). Most genes are found on the H strand. The L strand codes for a protein (ND subunit 6) and 8 tRNAs. From the H strand, two RNAs are transcribed, a short one for the rRNAs and a long one for mRNA and 14 tRNAs. A single transcript is made from the L strand. A 7 S RNA is transcribed in a counterclockwise manner close to the origin of replication (ORI), located between 11 and 12 o’clock on the circular structure.

 

B. Cooperation between mitochondrial and nuclear genome

Many mitochondrial proteins are aggregates of gene products of nuclear and mitochondrial genes. These gene products are transported into the mitochondria after nuclear transcription and cytoplasmic translation. In the mitochondria, they form functional proteins from subunits of mitochondrial and nuclear gene products. This explains why a number of mitochondrial genetic disorders show Mendelian inheritance, while purely mitochondrially determined disorders show exclusively maternal inheritance.

C. Evolutionary relationship of mitochondrial genomes

Mitochondria probably evolved from independent organisms that were integrated into cells. Similarities in structure and function between DNA in mitochondria, nuclear DNA, and DNA in chloroplasts suggest evolutionary relationships, in particular from chloroplasts to mitochondria, and from both to nuclear DNA of eukaryotic organisms.

 

 

 

Mitochondrial Disease

Mitochondria are ubiquitous organelles that are intimately involved in many cellular processes, but whose principal task is to provide the energy necessary for normal cell functioning and maintenance. Disruption of this energy supply can have devastating consequences for the cell, organ, and individual. Over the last two decades, mutations in both mitochondrial DNA (mtDNA) and nuclear DNA have been identified as causative in a number of well-characterized clinical syndromes, although for mtDNA mutations in particular, this relationship between genotype and phenotype is ofteot straightforward. Despite this, a number of epidemiological studies have been undertaken to assess the prevalence of mtDNA mutations and these have highlighted the impact that mtDNA disease has on both the community and individual families. Although there has been considerable improvement in the diagnosis of mitochondrial disorders, disappointingly this has not been matched by developments toward effective treatment. Nevertheless, our understanding of mitochondrial biology is gathering pace and progress in this area will be crucial to devising future treatment strategies. In addition to mitochondrial disease, evidence for a central role of mitochondria in other processes, such as aging and neurodegeneration, is slowly accumulating, although their role in cancer remains controversial. In this chapter, we discuss these issues and offer our own views based on our cumulative experience of investigating and managing these diseases over the last 20 years.

Mitochondria are intracellular organelles that play a critical role in cellular metabolism. Mitochondria contain the electron transport chain, which transfers electrons to oxygen by means of a process called oxidative phosphorylation. This process releases energy for the production of adenosine triphosphate (ATP) by forming a pH and electrical gradient (called the chemiosmotic gradient) across the inner mitochondrial membrane. In addition to oxidative phosphorylation, the mitochondria fulfill a number of other functions, including the following:

·  Make ATP for cellular energy

·  Metabolize fats, carbohydrates, and amino acids

·  Interconvert carbohydrates, fats, and amino acids

·  Synthesize some proteins

·  Reproduce themselves (replicate)

·  Participate in apoptosis

·  Make free radicals

Of these functions apoptosis is particularly important in development and disease. However, human disease may result from impairment of any of these functions.

Mitochondria are inherited from the mother, but not from the father. In the process of egg formation, there is thought to be a “bottleneck” in mitochondrial number, such that the unfertilized egg may have as few as 1,000 mitochondria. This number increases 100-fold after the ovum is fertilized. The mitochondria contain their own DNA, mitochondrial or mtDNA, and during development there may be selective amplification of some of these mtDNA molecules, leading to increases or decreases in the presence of mutated mtDNAs.

The Importance of the Electron Transport Chain

The origins of mitochondria are unknown, but the likely explanation, called the endosymbiont hypothesis, holds that they arose as free-living bacteria that colonized proto-eukaryotic cells, thereby establishing a symbiotic relationship. Primitive eukaryotic cells with intracellular mitochondria capable of metabolizing oxygen would have had an advantage in an oxygen-rich environment. The electron transport chain produces far more energy for each molecule of glucose consumed than is produced by anaerobic respiration. The oxidative phosphorylation process conducted by the mitochondria produces thirty-eight molecules of ATP, compared to two molecules of ATP produced by anaerobic glycolysis. Oxidative phosphorylation allows the conversion of toxic oxygen to water, a protective biological advantage.

A disadvantage of oxidative phosphorylation, however, is the formation of reactive oxygen species, such as singlet oxygen and hydroxyl radicals, which damage such cellular components as lipids, proteins, and DNA. A normally functioning electron transport chain produces reactive oxygen species from about 2 percent of the electrons that it transports. In disease states and in aging, larger quantities of reactive oxygen species are generated, and this may be a significant factor in cellular deterioration as well as a major contributor to the aging process.

Mitochondrial Genes and Disease

Mitochondrial DNA encodes approximately 3 percent of mitochondrial proteins. The relative contribution of the mitochondrial and nuclear genomes in coding for electron transport chain subunits is detailed in Figure 1. Human mtDNA contains 16,569 nucleotide bases and encodes thirteen polypeptides of the electron transport chain, twenty-two transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs). In addition, mtDNA has a control region (termed the D-loop), which contains considerable genetic variation. The D-loop forms the basis of forensic medicine DNA identification and has been very useful in the molecular anthropological study of human origins.

In 1988 the first human disease associated with mtDNA deletions was reported. These patients suffered from muscle and brain diseases with ragged red fiber muscle disease (myopathy), with or without progressive neurological deterioration. Ragged red fibers are muscle fibers, that have a disorganized structure and an excess of abnormal mitochondria and that stain red when treated with a histochemical stain called modified Gomori trichrome. In 1988 Kearns-Sayre syndrome, which primarily affects the muscles, heart, and brain, was found to be due to mtDNA deletions or duplications. About the same time, the maternally inherited disorder Leber’s hereditary optic atrophy was traced to point mutations in mitochondrial DNA encoding subunits of complex I of the electron transport chain.

Table 1

Organ or System Diseased		Symptoms
brain	stroke, seizures, dementia, ataxia, developmental delay
muscle	weakness, pain, fatigue
nerve	neuropathy
heart	cardiomyopathy, heart failure, heart block, arrhythmia
pancreas	diabetes, pancreatitis
eye	retinopathy, optic neuropathy
hearing	sensorineural deafness
kidney	renal failure
GI system	diarrhea, pseudo-obstruction, dysmotility

Table 1.

Mitochondrial diseases tend to affect multiple organ systems. The cells and organs most severely affected are those most heavily dependent on ATP, such as those listed in Table 1. Patients will frequently have multiple symptoms or signs, a circumstance that often causes confusion in diagnosis and treatment.

Classification

·         Mitochondrial myopathies

·         Diabetes mellitus and deafness (DAD)

• this combination at an early age can be due to mitochondrial disease

• Diabetes mellitus and deafness can also be found together for other reasons

• Leber’s hereditary optic neuropathy (LHON)

• visual loss beginning in young adulthood

• Wolff-Parkinson-White syndrome

• multiple sclerosis-type disease

• Leigh syndrome, subacute sclerosing encephalopathy

• after normal development the disease usually begins late in the first year of life, although onset may occur in adulthood

• a rapid decline in function occurs and is marked by seizures, altered states of consciousness, dementia, ventilatory failure

• Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP)

• progressive symptoms as described in the acronym

• dementia

• Myoneurogenic gastrointestinal encephalopathy (MNGIE)

• gastrointestinal pseudo-obstruction

• neuropathy

 

One of the more common presentations of mitochondrial disease in infants and young children is Leigh’s disease, first described by the pathologist Dennis Leigh in 1951. This progressive disease primarily affects the brain, with episodic deterioration that is often triggered by mild viral illnesses. Other organ systems are often involved, and there is often high blood or brain lactic acid as a result of a failure in oxidative metabolism (lactic acid is formed from glucose in the absence of oxygen). Complex I and IV defects are autosomal recessive diseases, with the culprit genes residing on the nuclear chromosomes. Complex V mutations are mtDNA inherited, and another 25 percent of cases are X-linked, due to pyruvate dehydrogenase deficiency

One of the most common mtDNA diseases seen is due to a single point mutation at position 3,243, with an adenine to guanine mutation in a tRNA leucine gene. Patients with this mutation may have phenotypes ranging from asymptomatic (that is, having no visible effects) to diabetes mellitus (with or without deafness). It is estimated that 1 to 2 percent of all diabetics have the A3243G mutation as the cause, affecting 200,000 people in the United States alone. The most severe phenotype to occur from this mutation has been given the acronym MELAS, for mitochondrial encephalomyopathy, with lactic acidosis and stroke-like episodes. The variability of disease phenotype or heterogeneity of disease due to mtDNA mutations arises in part because of variations in the amount of mutated mtDNA within different tissues. This mixture of wild type and mutant DNA within a cell is called heteroplasmy. In many mtDNA diseases, heteroplasmy changes over time, so that there is an increase in mutant DNA iondividing cells and tissues such as muscle, heart, and brain, with a decrease over time in rapidly dividing tissues such as bone marrow.

LEBER’S SYNDROME

(Leber Hereditary Optic Neuropathy ,Hereditary Optic Neuroretinopathy, LHON, Leber’s Disease, Leber’s Optic Atrophy, Leber’s Optic Neuropathy)

Clinical Diagnosis

Leber hereditary optic neuropathy (LHON) is characterized by bilateral, painless subacute visual failure that develops during young adult life. Males are approximately four times more likely to be affected than females.

Acute phase

Affected individuals are usually entirely asymptomatic until they develop visual blurring affecting the central visual field in one eye; similar symptoms appear in the other eye an average of eight weeks later. In an estimated 25% of cases, visual loss is bilateral at onset.

The ocular fundus may have a characteristic appearance that includes disk swelling, edema of the peripapillary nerve fiber layer, retinal telangiectasia, and an increased vascular tortuosity. The changes may be subtle; approximately 20% of affected individuals show no fundal abnormalities.

Visual acuity is severely reduced, to counting fingers or worse in most cases, and visual field testing (Goldmann perimetry or a similar technique) shows an enlarging central or centrocecal scotoma.

Atrophic phase.  After the acute phase, the optic discs become atrophic. Significant improvements in visual acuity are rare; in most individuals, vision remains severely impaired and within the legal requirement for blind registration.

Other findings.  The pathologic hallmark of LHON is the selective degeneration of the retinal ganglion cell layer and optic nerve.

Although visual failure is the defining feature in this mitochondrial disorder, cardiac arrhythmias and neurologic abnormalities such as postural tremor, peripheral neuropathy, nonspecific myopathy, and movement disorders have been reported to be more common in LHON as compared to controls . In addition, the association between all three primary LHON-causing mtDNA mutations and an MS-like illness among Caucasians, especially females, is well known .

Family history.  Affected individuals are often aware of other affected family members, but up to 40% have no family history [Harding et al 1995]. These most likely represent cases where family history is difficult to trace, given that de novo mutation is rare in LHON

Electrophysiologic studies (pattern electroretinogram and visual evoked potentials) confirm optic nerve dysfunction and the absence of retinal disease.

Cranial imaging is necessary to exclude other compressive, infiltrative, and inflammatory causes of a bilateral optic neuropathy. In individuals presenting with LHON, MRI is ofteormal but may reveal a high signal within the optic nerves, the latter probably representing slight edema or gliosis in the atrophic phase.

Testing

Biochemical studies.   Although the three primary LHON-causing mtDNA mutations all affect different respiratory chain complex I subunit genes, the mutations are not always associated with a respiratory chain abnormality that can be measured in vitro [Brown 1999]. The absence of a respiratory chain complex defect thus does not rule out the possibility of LHON.

In a small number of in vivo studies using phosphorus magnetic resonance spectroscopy, the most consistent defect of mitochondrial function was identified in individuals with the m.1778G>A mutation; it was not found among those with the m.3460G>A mutation (Table 1). A striking feature of all these biochemical studies is that none found a significant difference between affected and unaffected individuals with a disease-causing mutation. Balancing the current weight of evidence, LHON is associated with a respiratory chain defect that is more subtle than respiratory chain defects in other mitochondrial disorders and biochemical studies have been superseded by molecular genetic testing.

For current information on availability of genetic testing for disorders included in this section, see GeneTests Laboratory Directory. —ED.

If the ophthalmologic assessment (including an assessment of acuity, color vision, visual fields, and electrophysiology) and molecular genetic testing leave any uncertainty about the diagnosis of Leber hereditary optic neuropathy (LHON), further evaluation of the anterior visual pathways and brain with contrast MRI and lumbar puncture are appropriate to exclude other potentially treatable optic neuropathies.

Acute phase.   A wide range of non-genetic causes of bilateral visual failure must be excluded during the acute phase.

Atrophic phase.  If an individual is only seen at this stage, it can be difficult to exclude other possible causes of optic atrophy, especially if there is no clear maternal family history. In these cases, neuroimaging of the anterior visual pathways is mandatory while awaiting the results of molecular genetic testing.

 

LHON must also be distinguished from other causes of sporadic and inherited optic neuropathies such as deafness-dystonia-optic neuropathy (DDON). This disorder is characterized by prelingual or postlingual sensorineural hearing impairment in early childhood, slowly progressive dystonia or ataxia in the teens, slowly progressive decreased visual acuity from optic atrophy beginning at approximately age 20 years, and dementia beginning at approximately age 40 years. Psychiatric symptoms such as personality change and paranoia may appear in childhood and progress. The hearing impairment seems constant in age of onset and progression, whereas the neurologic, visual, and neuropsychiatric signs vary in degree of severity and rate of progression. Females may have mild hearing impairment and focal dystonia.

 

Management

Evaluations Following Initial Diagnosis

To establish the extent of disease in an individual diagnosed with Leber hereditary optic neuropathy (LHON), the following evaluations are recommended:

Measurement of best corrected visual acuity

Assessment of visual fields with static or kinetic perimetry

 

Treatment of Manifestations

Currently, no available treatment improves the final visual outcome in LHON.

Management of affected individuals is supportive and includes provision of visual aids and registration with the relevant social services.

ECG may reveal a pre-excitation syndrome in both affected and unaffected LHON carriers; such a finding does not necessitate further intervention in the absence of cardiac symptoms.

Agents/Circumstances to Avoid

Individuals harboring established LHON-causing mtDNA mutations are advised to avoid smoking and to moderate alcohol intake.

 

Mode of Inheritance

Leber hereditary optic neuropathy (LHON) is caused by mutations in mtDNA and is transmitted by maternal inheritance.

Risk to Family Members

Parents of a proband

The father of a proband is not at risk of having the disease-causing mtDNA mutation.

The mother of a proband usually has the mtDNA mutation and may or may not have symptoms.

In approximately 60% of cases, a history of visual loss affecting maternal relatives at a young age is present; up to 40% of individuals with LHON have no known family history of LHON. The explanation for these simplex cases may be that a detailed family history is not available or is unreliable, or that the proband has a de novo mtDNA mutation.

De novo mutations are assumed to be rare.

Sibs of a proband

The risk to sibs depends on the genetic status of the mother.

If the mother has the mtDNA mutation, all sibs are at risk of inheriting it.

Offspring of a proband

A male (affected or unaffected) with a primary LHON-causing mtDNA mutation cannot transmit the mutation to any of his offspring.

A female (affected or unaffected) with a primary LHON-causing mtDNA mutation transmits the mutation to all of her offspring.

The presence of the mtDNA mutation does not predict the occurrence, age of onset, severity, or the rate of progression of this typically adult-onset disease. See Risk Factors for Visual Loss for information regarding the risk to individuals with a primary LHON-causing mtDNA mutation of being affected.

If an affected female has heteroplasmy, she may transmit a low level of mutant mtDNA to her offspring, conferring a low disease risk

Other family members.   The risk to other family members depends on the genetic status of the proband’s mother. If the proband’s mother has a mtDNA mutation, her sibs and mother are also at risk.

Related Genetic Counseling Issues

Penetrance.   Genetic counseling for LHON is complicated by the gender- and age-dependent penetrance of the primary LHON-causing mtDNA mutations. Large studies have established accurate risks for the m.11778G>A and m.14484T>C mutations. Confirming the genetic status of an individual at risk for one of these mutations who is seeking counseling allows for an accurate estimation of the risks, based on established age- and gender-specific penetrance data (see Risk Factors for Visual Loss). Less data are available for the m.3460G>A mutation, and counseling for the other mutations requires cautious extrapolation.

Testing of at-risk asymptomatic adults.   Testing of at-risk asymptomatic adults for LHON is available using the same techniques described in Molecular Genetic Testing. Such testing is not useful in predicting age of onset, severity, or rate of progression in asymptomatic individuals. When testing at-risk individuals for LHON, an affected family member should be tested first to confirm the identification of the disease-causing mutation. The most important factors determining risk are gender and age. The presence of the mutation in leukocytes confers a lifetime risk (see Risk Factors for Visual Loss). For example, an 18-year-old male has a lifetime risk of approximately 50% for LHON after a positive test result. The risk declines with age but, because loss of sight can occur at any age, the risk never falls to zero. In large, multigenerational LHON pedigrees, these risks were known before the advent of molecular genetic testing. In smaller families it is important to confirm the genetic status because it is possible that the mutation is heteroplasmic in the affected individual or his mother, and it may not be present in every family member.

Testing for the disease-causing mutation in the absence of definite symptoms of the disease is predictive testing. At-risk asymptomatic adult family members may seek testing in order to make personal decisions regarding reproduction, financial matters, and career planning. Others may have different motivations including simply the “need to know.” Testing of asymptomatic at-risk adult family members usually involves pre-test interviews in which the motives for requesting the test, the individual’s knowledge of LHON, and the possible impact of positive and negative test results are assessed. Those seeking testing should be counseled about possible problems that they may encounter with regard to health, life, and disability insurance coverage, employment and educational discrimination, and changes in social and family interaction. Other issues to consider are implications for the at-risk status of other family members. Informed consent should be procured and records kept confidential.

Testing of at-risk individuals during childhood.  Consensus holds that individuals younger than age 18 years at risk for adult-onset disorders for which no treatment exists should not have testing in the absence of symptoms. The principal arguments against testing asymptomatic individuals during childhood are that it removes their choice to know or not know this information, it raises the possibility of stigmatization within the family and in other social settings, and it could have serious educational and career implications. Children who are symptomatic usually benefit from having a specific diagnosis established. (See also the National Society of Genetic Counselors resolution on genetic testing of children.)

Family planning.   The optimal time for determination of genetic risk is before pregnancy. Similarly, decisions about testing to determine the genetic status of at-risk asymptomatic family members are best made before pregnancy. It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk of developing LHON.

DNA banking.   DNA banking is the storage of DNA (typically extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. DNA banking is particularly relevant in situations in which the sensitivity of currently available testing is less than 100%. See for a list of laboratories offering DNA banking.

Prenatal Testing

Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at approximately 15-18 weeks’ gestation or chorionic villus sampling (CVS) at approximately ten to 12 weeks’ gestation. The mtDNA mutation in the mother must be identified before prenatal testing can be performed.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Accurate interpretation of a positive prenatal test result is difficult for the following reasons:

Because of mitotic segregation, the mtDNA mutational load in amniocytes and chorionic villi is unlikely to correspond to that of other fetal or adult tissues.

The presence of the mtDNA mutation does not predict the occurrence, age of onset, severity, or rate of progression of this typically adult-onset disease. See Risk Factors for Visual Loss for information regarding the risk to individuals with a primary LHON-causing mtDNA mutation of being affected.

 

MERRF syndrome

Disease characteristics.

The clinical diagnosis of MERRF (myoclonic epilepsy with ragged red fibers) is based on the following four “canonical” features:

 

o                   Myoclonus

o                   Generalized epilepsy

o                   Ataxia

o                   Ragged red fibers (RRF) in the muscle biopsy

o                   Additional frequent manifestations include the following:

o                   Sensorineural hearing loss

o                   Myopathy

o                   Peripheral neuropathy

o                   Dementia

o                   Short stature

o                   Exercise intolerance

o                   Optic atrophy

o                   Less common clinical signs (seen in <50% of affected individuals) include the following:

o                   Cardiomyopathy

o                   Pigmentary retinopathy

o                   Pyramidal signs

o                   Ophthalmoparesis

o                   Multiple lipomas

 

 

 

 

Diagnosis/testing.

 The clinical diagnosis of MERRF is based on the following four “canonical” features: myoclonus, generalized epilepsy, ataxia, and ragged red fibers (RRF) in the muscle biopsy. The mitochondrial DNA (mtDNA) gene MT-TK encoding tRNALys is the gene most commonly associated with MERRF. The most common mutation, present in over 80% of affected individuals with typical findings, is an A-to-G transition at nucleotide 8344 (m.8344A>G). Mutations are usually present in all tissues and are conveniently detected in mtDNA from blood leukocytes. However, the occurrence of “heteroplasmy” in disorders of mtDNA can result in varying tissue distribution of mutated mtDNA. Hence, in individuals having few symptoms consistent with MERRF or in asymptomatic maternal relatives of an affected individual, the pathogenic mutation may be undetectable in mtDNA from leukocytes and may only be detected in other tissues, such as cultured skin fibroblasts, urinary sediment, oral mucosa, hair follicles, or, most reliably, skeletal muscle.

Management.

Treatment of manifestations: conventional antiepileptic drugs (AEDs) for seizures; physical therapy to improve any impaired motor function; aerobic exercise; standard pharmacologic therapy for cardiac symptoms. Levetiracetam, clonazepam, zonisamide, and valproic acid (VPA) have been used to treat myoclonic epilepsy; however, VPA may cause secondary carnitine deficiency and should be avoided or used with L-carnitine supplementation. Other: Coenzyme Q10 (100 mg 3x/day) and L-carnitine (1000 mg 3x/day) are often used in hopes of improving mitochondrial function.

Genetic counseling.

 MERRF is caused by mutations in mtDNA and is transmitted by maternal inheritance. The father of a proband is not at risk for having the disease-causing mtDNA mutation. The mother of a proband usually has the mtDNA mutation and may or may not have symptoms. A male with an mtDNA mutation cannot transmit the mutation to any of his offspring. A female with the mutation (whether affected or unaffected) transmits the mutation to all of her offspring. Prenatal diagnosis for MERRF is possible if an mtDNA mutation has been detected in the mother. However, because the mutational load in the mother’s tissues and in the fetal tissues sampled (i.e., amniocytes and chorionic villi) may not correspond to that of other fetal tissues and because the mutational load in tissues sampled prenatally may shift in utero or after birth secondary to random mitotic segregation, prediction of the phenotype from prenatal studies is not possible.

 

MELAS Syndrome

 

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome is a progressive neurodegenerative disorder. Patients may present sporadically or as members of maternal pedigrees with a wide variety of clinical presentations. The typical presentation of patients with MELAS syndrome includes features that comprise the name of the disorder, such as mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes. Other features, such as seizures, diabetes mellitus, hearing loss, cardiac disease, short stature, endocrinopathies, exercise intolerance, and neuropsychiatric dysfunction are clearly part of the disorder.

Pathophysiology

Strokelike episodes and mitochondrial myopathy characterize MELAS syndrome. Multisystemic organ involvement is seen, including the CNS, skeletal muscle, eye, cardiac muscle, and, more rarely, the GI and renal systems.

Approximately 80% of patients with the clinical characteristics of MELAS syndrome have a heteroplasmic A-to-G point mutation in the dihydrouridine loop of the transfer RNA (tRNA)^{Leu (UUR)} gene at base pair (bp) 3243 (ie, 3243 A → G mutation).¹ However, other mitochondrial DNA (mtDNA) mutations are observed, including the m.3244 G → A, m.3258 T → C, m.3271 T → C, and m.3291 T → C in the mitochondrial tRNA^Leu(UUR) gene.

The pathogenesis of the strokelike episodes in MELAS syndrome has not been completely elucidated. These metabolic strokelike episodes may be nonvascular and due to transient oxidative phosphorylation (OXPHOS) dysfunction within the brain parenchyma. A mitochondrial angiopathy of small vessel is responsible for contrast enhancement of affected regions and mitochondrial abnormalities of endothelial cells and smooth muscle cells of blood vessels. The multisystem dysfunction in patients with MELAS syndrome may be due to both parenchymal and vascular OXPHOS defects. Increased production of free radicals in association with an OXPHOS defect leading to vasoconstriction may offset the effect of potent vasodilators (eg, nitric oxide).

The unusual strokelike episodes and higher morbidity observed in MELAS syndrome may be secondary to alterations iitric oxide homeostasis that cause microvascular damage. Nitric oxide can bind the cytochrome c oxidase–positive sites in the blood vessels present in the CNS, displacing heme-bound oxygen and resulting in decreased oxygen availability in the surrounding tissue and decreased free nitric oxide. Furthermore, coupling of the vascular mitochondrial dysfunction with cortical spreading depression might underlie the selective distribution of ischemic lesions in the posterior cortex in these subjects.   

Mutations in this disorder affect mitochondrial tRNA function, leading to the disruption of the global process of intramitochondrial protein synthesis. Measurements of respiratory enzyme activities in intact mitochondria have revealed that more than one half of the patients with MELAS syndrome may have complex I or complex I + IV deficiency. A close relationship is apparent between MELAS and complex I deficiency. The decreased protein synthesis may ultimately lead to the observed decrease in respiratory chain activity by reduced translation of UUG-rich genes such as ND6 (component of complex I).

In addition, studies revealed that the 3243 A → G mutation produces a severe combined respiratory chain defect in myoblasts, with almost complete lack of assembly of complex I, IV, and V, and a slight decrease of assembled complex III. This assembly defect occurs despite a modest reduction in the overall rate of mitochondrial protein synthesis. Translation of some polypeptides is decreased, and evidence of amino acid misincorporation is noted in others.

Frequency

United States

No estimates concerning the prevalence of the common MELAS mutation are available for the North American population; however, the syndrome has been observed to be less frequent in blacks.

International

The first assessment of the epidemiology of mitochondrial disorders found a prevalence of more than 10.2 per 100,000 for the m.3243A → G mutation in the adult Finnish population. If the assumption is made that all first-degree maternal relatives of a verified mutation carrier also harbor the mutation, prevalence increases to more than 16.3 per 100,000. This high prevalence suggests that mitochondrial disorders may constitute one of the largest diagnostic categories of neurogenetic diseases among adults. In Northern England, the prevalence of this mutation in the adult population has been determined to be approximately 1 per 13,000.

Mortality/Morbidity

The progressive disorder has a high morbidity and mortality. The encephalomyopathy, associated with strokelike episodes followed by hemiplegia and hemianopia, is severe. Focal and general convulsions may occur in association with these episodes.

Other abnormalities that may be observed are ventricular dilatation, cortical atrophy, and basal ganglia calcification. Mental deterioration usually progresses after repeated episodic attacks. Psychiatric abnormalities and cognitive decline (eg, altered mental status, schizophrenia) may accompany the strokelike episodes. Bipolar disorder is another psychiatric abnormality observed in MELAS syndrome. Autism spectrum disorders (ASDs) with or without additional neurological features can be early presentations of the m.3243 A → G mutation. Myopathy may be debilitating. The encephalopathy may progress to dementia; eventually, the clinical course rapidly declines, leading to severe disability and premature death.

Another cause of high mortality is the less common feature of cardiac involvement, which can include hypertrophic cardiomyopathy, hypertension, and conduction abnormalities, such as atrioventricular blocks, long QT syndrome, or Wolff-Parkinson-White syndrome. Subjects with MELAS syndrome were found to have increased ascending aortic stiffness and enlarged aortic dimensions suggesting vascular remodeling. Aortic root dissection was found in one patient with MELAS syndrome.³ Some patients may develop Leigh syndrome (ie, subacute necrotizing encephalopathy). Patients may develop renal failure due to focal segmental glomerulosclerosis.

More rarely, these patients may exhibit severe GI dysmotility and endocrine dysfunction, including hypothyroidism and hyperthyroidism.

Race

No predilection for a particular ethnic group is noted.

Sex

No sexual predilection is present.

Age

In many patients with MELAS syndrome, presentation occurs with the first strokelike episode, usually when an individual is aged 4-15 years. Less often, onset of disease may occur in infancy with delayed developmental milestones and learning disability. One presentation of the disorder was reported in a 4-month-old infant.

History

• Onset of the disorder may be myopathic with weakness, easy fatigability, and exercise intolerance.

• Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome onset may occur early in infancy with a history of developmental delay and learning disabilities. Developmental delay, learning disability, or attention deficit disorder is primarily found in patients prior to the development of the first stroke. An encephalopathic picture that is progressive and leads to dementia may be present. Patients may be apathetic. The level of cognitive functioning worsens over time by Karnofsky score in fully symptomatic patients.

• Failure to thrive may be the presenting feature in some patients with MELAS syndrome.

• Strokelike episodes are the hallmark feature of this disorder. Initially, episodes may manifest with vomiting and headache that may last several days. These patients may also experience episodes of seizures and visual abnormalities followed by hemiplegia. Seizure types may be tonic-clonic or myoclonic.

• Migraine or migrainelike headaches observed in these patients may also reflect the strokelike episodes. Pedigrees of patients with classic MELAS syndrome identify many members whose only manifestations are migraine headaches.

• Patients may have visual complaints due to ophthalmoplegia, and they may experience blindness because of optic atrophy and difficulties with night vision due to pigmentary retinopathy.

• Some patients may experience hearing loss, which may accompany diabetes. It may be observed in association with the classic disorder of MELAS syndrome.

• Polydipsia and polyuria may be the presenting signs of diabetes; diabetes appears to be the most common manifestation of MELAS syndrome. Usually, type 2 diabetes is described in individuals with MELAS syndrome, although type 1 (formerly termed insulin-dependent diabetes) may also be observed. Guidelines for diagnosis and management of type 2 diabetes have been established.⁴

• Palpitations and shortness of breath may be present in some patients with MELAS syndrome secondary to cardiac conduction abnormalities, such as Wolff-Parkinson-White syndrome. Patients may experience shortness of breath secondary to cardiomyopathy, which is usually of the hypertrophic type; however, dilated cardiomyopathy has also been described.

• Acute onset of GI manifestations (eg, acute onset of abdominal pain) may reflect pancreatitis, ischemic colitis, and intestinal obstruction.

• Numbness, tingling sensation, and pain in the extremities can be manifestations of peripheral neuropathy.

• Psychiatric disorders (eg, depression, bipolar disorder) have been associated with the m.3243 A → G mutation. Dementia has been another clinical manifestation. Moreover, autism spectrum disorders (ASD) have been associated with the 3243 A → G mutation.

• Patients may develop features of hypothyroidism and hyperthyroidism

• Some patients may develop apnea and an ataxic gait in association with neuroradiologic features of MELAS syndrome.

• Oliguria can be associated with MELAS syndrome and may indicate the onset of nephrotic syndrome.

• Patients with MELAS syndrome may have functional vascular involvement. Aortic root dissection has been reported in one patient with MELAS syndrome.

Physical

• Patients with MELAS syndrome may exhibit hypertension.

• Myopathy presents with hypotonia and weakness. Proximal muscles tend to be more involved than distal muscles. Musculature is thin, and patients may present with a myopathic face.

• Strokelike episodes may present with convulsions, visual abnormalities, numbness, hemiplegia, and aphasia. Episodes may be followed by transient hemiplegia or hemianopia, which lasts a few hours to several weeks.

• Additional features oeurologic examination may include ataxia, tremor, myoclonus, dystonia, visual disturbances, and cortical blindness. Some patients may present with ophthalmoplegia and ptosis.

• On ophthalmologic examination, patients have presented with pigmentary retinopathy.

• Sensorineural deafness has been reported as part of the disorder in approximately 25% of patients with MELAS syndrome.

• Cardiomyopathy with signs of congestive heart failure (CHF) may also be observed upon physical examination.

• Skin manifestations of cutaneous purpura, hirsutism, and a scaly, pruritic, diffuse erythema with reticular pigmentation may be observed in patients with MELAS syndrome.

• Short stature may be the first manifestation of MELAS syndrome in many patients.

Causes

• MELAS syndrome has been associated with at least 6 different point mutations, 4 of which are located in the same gene, the tRNA^{Leu (UUR)} gene. The most common mutation, found in 80% of individuals with MELAS syndrome, is an A → G transition at nucleotide (nt) 3243 in the tRNA^{Leu (UUR)} gene. An additional 7.5% have a heteroplasmic T → C point mutation at bp 3271 in the terminal nucleotide pair of the anticodon stem of the tRNA^{Leu (UUR)} gene. Moreover, a MELAS phenotype has been observed associated with an m.13513G → A mutation in the ND5 gene and in POLG deficiency.   

• These mutations are heteroplasmic, which reflects the different percentages of mutated mtDNA present in different tissues. Variable heteroplasmy among individuals affected with MELAS syndrome reflects variable segregation in the ovum. Mutations in tRNA^Lys may be expected to have an important effect on translation and protein synthesis in mitochondria. The MELAS disorder–associated human mitochondrial tRNA^{Leu (UUR)} mutation causes aminoacylation deficiency and a concomitant defect in translation initiation.

• Abnormal calcium homeostasis resulting ieuronal injury has been suggested as another mechanism contributing to the CNS involvement observed in MELAS syndrome.

• Patients with MELAS syndrome have been found to have a marked decrease in the activity of complex I. The major effects observed secondary to nt 3243 and nt 3271 mutations have been a reduction in protein synthesis and the activity of complex I. These effects have been demonstrated through studying cybrids in which human cell lines without mtDNA are fused with exogenous mitochondria containing 0-100% of the common m.3243 mutation. Cybrids with more than 95% of mutant DNA had decreased rates of synthesis of mitochondrial proteins, leading to respiratory chain defects.

Other Problems to Be Considered

Sensorineural hearing loss
Peripheral neuropathy
Rhabdomyolysis
Intestinal pseudoobstruction
Myoclonic epilepsy and ragged red fiber disease
Neurodegeneration, ataxia, and retinitis pigmentosa
Primary mtDNA depletion syndrome
Disorders of pyruvate metabolism

Laboratory Studies

• Serum lactic acid, serum pyruvic acid, cerebrospinal fluid (CSF) lactic acid, and CSF pyruvic acid  

• Lactic acidosis is an important feature of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome.

 

 [ CLOSE WINDOW ]

Pathophysiologic classification of lactic acidosis.

• In general, lactic acidosis does not lead to systemic metabolic acidosis, and it may be absent in patients with impressive CNS involvement.

• In some individuals with MELAS syndrome, lactic acid levels may be normal in blood but elevated in CSF.

• In respiratory chain defects, the ratio between lactate and pyruvate is high.

• Serum creatine kinase levels

• The levels of serum creatine kinase are mildly to moderately increased in some patients with MELAS syndrome.

• Levels tend to increase during and immediately after episodes.

• Respiratory chain enzyme activities in skeletal muscle  

• If a muscle biopsy is performed to pursue a diagnostic evaluation, then test respiratory chain enzyme activities.

• Patients with MELAS syndrome have been found to have marked deficiency in complex I activity of the respiratory chain.

• Some patients with the disorder have a combined deficiency of complex I and complex IV.

• Mitochondrial DNA mutation analysis on blood, skeletal muscle, hair follicles, buccal mucosa, and urinary sediment

• Individuals with more severe clinical manifestations of MELAS syndrome generally have greater than 80% mutant mtDNA in stable tissues such as muscle.

• In rapidly dividing cells, such as the components of the hematopoietic lineages, the m.3243 A → G mutation may segregate to extremely low levels, making genetic diagnosis from blood difficult. The percentage of the mutation decreases progressively in DNA isolated from blood. The mutant load isolated from blood is neither useful for prognosis nor for functional assessment.

• Urinary sediment, followed by skin fibroblasts and buccal mucosa, are the accessible tissues of choice because they are easy to access and the mutation load is higher than that found in blood.

• If the diagnosis is still suspected after normal mtDNA mutation analysis results in these tissues, a skeletal muscle biopsy is required to confirm or rule out the presence of the mutation.

Imaging Studies

• CT scan or MRI of the brain

• CT scan or MRI of the brain following a strokelike episode reveals a lucency that is consistent with infarction.

• Later, cerebral atrophy and calcifications may be observed on brain imaging studies.

• Patients with MELAS syndrome who have a presentation similar to Leigh syndrome may have calcifications in the basal ganglia.

• Positron emission tomography (PET) studies

• PET studies may reveal a reduced cerebral metabolic rate for oxygen.

• Increased cerebral blood flow in cortical regions may be observed.

• PET may demonstrate preservation of the cerebral metabolic rate for glucose.

• Single-photon emission CT studies

• Single-photon emission computed tomography (SPECT) studies can ascertain strokes in individuals with MELAS syndrome using a tracer, N -isopropyl-p-[123-I]-iodoamphetamine.

• The tracer accumulates in the parietooccipital region, and it can delineate the extent of the lesion. SPECT studies are used to monitor the evolution of the disease.

• Proton magnetic resonance spectroscopy (¹ H-MRS): This is used to identify metabolic abnormalities, including the lactate-to-creatine ratio in either muscle or brain and the decreased CNS N -acetylaspartate–to–creatine ratio in regions of stroke. With this technique, elevated regions of lactate have been detected while serum levels are normal.

• Echocardiography: This is useful to evaluate for hypertrophic and dilated cardiomyopathy and aortic root dimensions; however, cardiomyopathy is not a common feature in individuals with MELAS syndrome.

Other Tests

• EEG

• EEG findings are usually abnormal.

• Epileptiform spike discharges are usually present.

• ECG

• ECG is used to look for conduction abnormalities with ventricular arrhythmias.

• ECG can identify presymptomatic cardiac involvement, preexcitation syndromes, and cardiac conduction block.

Procedures

• Consider performing a muscle biopsy if MELAS syndrome is suspected and if the mtDNA mutation analysis in blood and other accessible tissues provides unremarkable results.

• In rapidly dividing cell lines, the mutations may segregate to low levels, making genetic diagnosis from blood difficult.

Histologic Findings

• In muscle biopsies stained with hematoxylin and eosin, variation is observed in type 1 and type 2 fiber sizes, representing myopathic changes.

• Ragged red fibers are the hallmark of MELAS syndrome. The ragged red fibers stain brilliant red with occasional cytoplasmic bodies with trichrome stain. Ragged red fibers usually stain positive with cytochrome oxydase stain.

• Staining with periodic acid-Schiff, nicotinamide adenine dinucleotide (NADH) dehydrogenase tetrazolium reductase, or for succinic dehydrogenase demonstrates increased subsarcolemmal activity. This mitochondrial proliferation has also been observed in blood vessels and is determined using a stain for succinate dehydrogenase.

• Electron microscopy demonstrates an increase iumber and size of mitochondria, some with paracrystalline bodies.

Medical Care

• Evaluation for  mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome may be performed on an outpatient basis if the patient is stable.

• Evaluation may consist of determining levels of serum lactate and serum pyruvate, mtDNA mutation studies on blood, and brain imaging studies (eg, head CT scan, brain MRI, brain¹ H-MRS).

• Muscle biopsy for mitochondrial enzymes and DNA mutation analysis can be performed as an elective procedure for which the patient is admitted to the hospital.

• In incidents of acute decompensation, perform inpatient studies in the acute phase and following stabilization of the patient.

• Various supportive measures are available, although no controlled trial has proven efficacy. Long-term benefits of dietary manipulations are unknown. Improvements in some patients may be related to improved nutritional status and hydration.

• Treatment with coenzyme CoQ10 has been helpful in some patients with MELAS syndrome. No adverse effects have been reported from its administration.

• Menadione (vitamin K-3), phylloquinone (vitamin K-1), and ascorbate have been used to donate electrons to cytochrome c.

• Idebenone has also been used to treat this condition, and improvements in clinical and metabolic abnormalities have been reported.

• Riboflavin has been reported to improve the function of a patient with complex I deficiency and the m.3250 T→C mutation.

• Nicotinamide has been used because complex I accepts electrons from nicotinamide adenine dinucleotide (NADH) and ultimately transfers electrons to CoQ10.

• Dichloroacetate is another compound used with these agents since levels of lactate are lowered in plasma and cerebrospinal fluid (CSF); patients reportedly may respond in a favorable manner. Sensory neuropathy may result after extended use of this drug.

• Sodium succinate has been used, and a patient with MELAS syndrome reportedly had fewer strokelike episodes with its use; however, sodium succinate is not the standard of care. Further investigation is necessary.

• Creatine monohydrate has also been used, and an increase in muscle strength in high-intensity anaerobic and aerobic activities has been reported.

• The administration of L-arginine during the acute and interictal periods may represent a potential new therapy for this syndrome to reduce brain damage due to impaired vasodilation in intracerebral arteries owing to nitric oxide depletion.

Consultations

• Geneticist

• Neurologist (to evaluate patient for strokelike episodes)

• Cardiologist (for evaluation of cardiomyopathy, arrhythmias and hypertension)

• Nephrologist (to evaluate for the onset of nephrotic syndrome)

• Ophthalmologist (to evaluate for pigmentary retinopathy)

• Endocrinologist (to evaluate for endocrine dysfunctions such as diabetes mellitus, hypothyroidism, hyperthyroidism and hypoparathyroidism)

• Psychiatrist (to evaluate for affective disorders)

• Neuropsychologist (to evaluate for autism spectrum disorder [ASD])

Diet

• The effect of dietary manipulation is not completely known, and the efficacy of dietary supplements is unproven.

• Dicarboxylic aciduria and secondary impairment of long-chain fatty acid oxidation (LCFAO) may occur in mitochondrial disorders.

• Improvement observed in many patients is probably related to improved nutrition.

Activity

• In patients with mitochondrial myopathies, moderate treadmill training may result in improvement of aerobic capacity and a drop in resting lactate and postexercise lactate levels.

• Concentric exercise training may also play an important role because after a short period of concentric exercise training a remarkable increase reportedly occurs in the ratio of wild type–to–mutant mtDNAs and in the proportion of muscle fibers with normal respiratory chain activity.

For individuals with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome and for those with other oxidative phosphorylation (OXPHOS) disorders, metabolic therapies are administered to increase the production of adenosine triphosphate (ATP) and to slow or arrest the deterioration of this condition and other mitochondrial encephalomyopathies. Metabolic therapies used for the management of MELAS syndrome include carnitine, CoQ10, phylloquinone, menadione, ascorbate (ie, ascorbic acid), riboflavin, nicotinamide, creatine monohydrate, idebenone, succinate, and dichloroacetate. However, assessment of the efficacy of these compounds is far from complete, and efficacy is believed to be limited to individual cases.

Treatment with CoQ10 has been helpful in some patients with MELAS syndrome. No adverse effects have been reported from its administration. Menadione (vitamin K-3), phylloquinone (vitamin K-1), and ascorbate have been used to donate electrons to cytochrome c. Idebenone has also been used to treat this condition, and improvements in clinical and metabolic abnormalities have been reported. Riboflavin has been reported to improve the function of a patient with complex I deficiency and the m.3250 T→C mutation. Nicotinamide has been used because complex I accepts electrons from nicotinamide adenine dinucleotide (NADH) and ultimately transfers electrons to Q10. Dichloroacetate is another compound used with these agents, because levels of lactate are lowered in plasma and cerebrospinal fluid (CSF). Patients reportedly may respond in a favorable manner.

A patient with MELAS syndrome reportedly had fewer strokelike episodes with the use of sodium succinate; however, sodium succinate is not the standard of care, and further investigation is necessary. An increase in muscle strength in high-intensity anaerobic and aerobic activities has been reported with the administration of creatine monohydrate.

Arginine administration during the acute and interictal periods of the strokelike episodes of the MELAS syndrome may represent a potential new therapy to reduce brain damage due to mitochondrial dysfunction, and is one of the most promising therapies to date. Based on the hypothesis that the strokelike episodes in MELAS syndrome are triggered by impaired vasodilation in the intracerebral arteries due to decreased levels of circulating NO, elevation of arginine and NO levels may ameliorate this effect. In addition, L-arginine may modulate excitation by neurotransmitters at nerve endings and such effects might contribute to alleviation of strokelike symptoms in MELAS syndrome. Patients with MELAS may have less chance of having strokelike episodes by improving their endothelial function with oral supplementation of L-arginine.

 

Vitamins and dietary supplements

Vitamins are organic substances the body requires in small amounts for various metabolic processes. Vitamins may be synthesized in small or insufficient amounts in the body or not synthesized at all, thus requiring supplementation. Some case reports using dietary supplements have reported an improvement in patient symptoms.

Further Inpatient Care

• Admit for medical management of strokelike episodes and seizures.

• Admit for metabolic decompensation or signs of diabetic ketoacidosis. Diabetes appears to be the most common manifestation of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) syndrome.

• Admit for signs of cardiac arrhythmia (Wolff-Parkinson-White syndrome), hypertension, impending aortic root dissection, or congestive heart failure (CHF) associated with hypertrophic or dilated cardiomyopathy.

• Admit for signs of nephrotic syndrome that may present in association with focal segmental glomerulosclerosis.

• Admit if a sign of acute abdomen is present; acute abdomen may be an indication of pancreatitis.

Further Outpatient Care

• Carefully monitor the progress of the encephalomyopathy and sequelae.

• Neurodevelopmental testing is appropriate because progressive intellectual deterioration follows stroke-like episodes of MELAS syndrome. Neuropsychological evaluation is appropriate for presence of autism spectrum disorder (ASD).

• Monitor growth curves because mitochondrial disorders such as MELAS syndrome are associated with short stature or failure to thrive.

• Refer the patient to an ophthalmologist to monitor for pigmentary degeneration of the retina, which may be similar to that observed in patients with neuropathy, ataxia, and retinitis pigmentosa syndrome. Closely monitor signs (eg, ophthalmoplegia, ptosis).

• Carefully monitor individuals with MELAS syndrome for hearing loss with a hearing evaluation, including distortion product otoacoustic emissions and auditory brainstem evoked responses.

• Carefully monitor patients for cardiomyopathy and measure Z-score for aortic root diameter with echocardiography. Request an ECG as a baseline study to monitor for conduction defects, even if patients are asymptomatic.

• Carefully monitor patients for type 2 diabetes, hypothyroidism, hyperthyroidism, and parathyroid dysfunction.

• Carefully monitor patients for the persistence of lactic acidosis.

• ¹ H-MRS of the brain may be used to monitor potential therapeutic efficacy if increased permeability of the blood-brain barrier is a concern.

Inpatient & Outpatient Medications

• Medications include the following:

• Compounds that may increase ATP production or transfer of electrons (eg, ascorbate, riboflavin, CoQ10, vitamins K-1 and K-3, nicotinamide, creatine monohydrate)

• Compounds that can be used to prevent a possible secondary carnitine deficiency or secondary dysfunction of fatty acid oxidation (eg, carnitine)

• Compounds that can be used to prevent or ameliorate the progression of stroke-like episodes (eg, L-arginine): L-arginine could modulate mitochondrial energy metabolism by inhibiting glutamate uptake into mitochondria and decreasing neurotoxicity associated with nitric oxide-mediated mitochondrial dysfunction.  

• Compounds that may be used to treat lactic acidosis (eg, dichloroacetate)

• Dichloroacetate stimulates pyruvate dehydrogenase function by inhibiting pyruvate dehydrogenase kinase, the enzyme that normally phosphorylates and inactivates pyruvate dehydrogenase. Therefore, in conditions that result in the accumulation of lactate and alanine, activation of pyruvate dehydrogenase decreases the release of these compounds from peripheral tissues and enhances their oxidative metabolism by the liver.

• This medication has been used to treat lactic acidosis in adult and pediatric patients. Anecdotal reports detail successful treatment in patients with MELAS syndrome. Dichloroacetate has been administered orally at doses of 12.5-100 mg/kg/d. This medication is available only under research protocols in the United States.

• If seizures have developed as part of the condition, do not use valproic acid as an anticonvulsant, since incidents of pancreatitis following valproate administration have occurred and valproic acid has been associated with mitochondrial toxicity.

• Use phenobarbital with caution, because the drug has demonstrated inhibition of the respiratory chain in vitro.

Transfer

• Transfer to a tertiary care center may be required to better coordinate the diagnostic evaluation to include the following:

• Muscle biopsy

• Evaluation for mitochondrial enzyme defects

• Analysis of mtDNA mutation

• If diagnosis is already known and the patient has been stabilized, transfer may be required for better management of complications such as the following:

• Pancreatitis

• Cardiac arrhythmias

• Cardiomyopathy

• Ketoacidosis

• Stroke-like episodes

Deterrence/Prevention

• If conditions such as cardiomyopathy are present, restrict exercise.

• Although the long-term effects of dietary manipulations are unknown, ensure good nutritional status, good hydration, and avoidance of fasting as part of a supportive plan.

• A mild degree of aerobic activity may lead to an improvement of aerobic capacity. Restrict strenuous exercise because of the possible complication of rhabdomyolysis.

• Information on the therapeutic efficacy of reported compounds used as nutritional supplements are limited; however, most do not have any serious adverse effects. Nutritional supplements may help to prevent further deterioration in some individuals; however, further research is warranted.

Complications

• Failure to thrive and short stature

• Progressive intellectual deterioration and decline that eventually may lead to dementia

• Psychosis with depression, schizophrenia, or bipolar disorder

• Autism spectrum disorders (ASDs)

• Sensorineural hearing loss

• Endocrine dysfunction with hypogonadism, diabetes, hypoparathyroidism, hypothyroidism, and hyperthyroidism

• CHF from cardiomyopathy and sudden death from conduction defects

• Visual difficulties related to pigmentary degeneration of the retina or cortical blindness as one of the sequelae of progressive cortical atrophy and strokelike episodes

• End-stage renal failure as a complication of focal segmental glomerulosclerosis

• Acute renal failure secondary to rhabdomyolysis

• GI dysfunction secondary to intestinal pseudoobstruction or pancreatitis

• Aortic root dissection (reported in one kindred; requires further studies to evaluate the prevalence)

Prognosis

• MELAS syndrome widely varies in presentation; however, patients in general tend to have a poor prognosis and outcome.

• The encephalomyopathy tends to be severe and progressive to dementia. The patient with MELAS syndrome may end up in a state of cachexia.

• Currently, no therapies have proven efficacy.

Pearson Syndrome

 

Background

In 1979, Pearson et al described a previously unrecognized, often fatal disorder of infants with transfusion-dependent sideroblastic anemia, vacuolization of hematopoietic precursors, and exocrine pancreatic insufficiency. The large deletions of the mitochondrial genome that cause the disorder were discovered a decade later.

Pearson syndrome is currently recognized as a rare, multisystemic, mitochondrial cytopathy. Its features are refractory sideroblastic anemia, pancytopenia, defective oxidative phosphorylation, exocrine pancreatic insufficiency, and variable hepatic, renal, and endocrine failure. Death often occurs in infancy or early childhood due to infection or metabolic crisis. Patients may recover from the refractory anemia. Older survivors have Kearns-Sayre syndrome (KSS), which is a mitochondropathy characterized by progressive external ophthalmoplegia and weakness of skeletal muscle.

Pathophysiology

Mitochondropathies

The mitochondropathies comprise several diverse, overlapping syndromes caused by mutations of mitochondrial DNA. Pearson syndrome is a specific clinical subset of these syndromes that in which involvement of the bone marrow and exocrine pancreas is prominent. The pathogenesis of Pearson syndrome is complex and not well understood. Deletions of certain components of the electron transport chain, encoded by mitochondrial DNA, cause a defect in cellular oxidative metabolism. Certain transfer RNAs (tRNAs) may also be deleted, and their deletion impairs the translation of messenger RNAs (mRNAs) to proteins. Abnormal metabolism of iron, evidenced by sideroblastosis and hemosiderosis, may also be a key feature (see Image 3). These defects cause cellular injury in target tissues.

Other mitochondropathies, such as KSS and the mitochondrial myopathies, have deletions of mitochondrial DNA that may be similar or identical to those detected in Pearson syndrome. How similar abnormalities of mitochondrial DNA cause such diverse disorders is not well understood. The distinct phenotypes are probably the result of differences in the amount and in the tissue-specific distribution of abnormal mitochondrial DNA, the evolution of this distribution over time, and the effects of tissue-specific nuclear modifier genes.

Defining features of Pearson syndrome

The first defining feature of Pearson syndrome is marrow failure. Macrocytic sideroblastic anemia occurs with the characteristic vacuolation of hematopoietic precursors (see Images 1-2). The anemia is refractory, and patients may be transfusion dependent. Neutropenia and thrombocytopenia may also be present.

The second defining feature of Pearson syndrome is dysfunction of the exocrine pancreas due to fibrosis and acinar atrophy. The result is malabsorption and chronic diarrhea.

Another cardinal feature of Pearson syndrome is persistent or intermittent lactic acidemia, which is caused by a defect in oxidative phosphorylation.

Other organ systems are affected in various ways. Hepatic involvement may cause increases in transaminase, bilirubin, and lipid levels, as well as in steatosis. Some patients develop hepatic failure. Renal involvement is common and manifests as a tubulopathy, such as Fanconi syndrome. Endocrinologic disturbances, such as growth hormone deficiency, hypothyroidism, and hypoparathyroidism, are relatively uncommon. The endocrine pancreas usually remains functional; however, a few patients develop diabetes mellitus. Splenic atrophy and impaired cardiac function have also been reported.

Failure to thrive is common. Several factors are likely contributory. Such factors include a defect in cellular metabolic energy, malabsorption due to exocrine pancreatic failure, hepatic and renal insufficiency, and, perhaps, concomitant endocrinologic abnormalities.

Frequency

United States

Pearson syndrome is rare. Approximately 60 cases have been reported worldwide.

Mortality/Morbidity

Pearson syndrome is often fatal in infancy or early childhood. The usual causes of death are bacterial sepsis due to neutropenia, metabolic crisis, and hepatic failure.

Race

All races can be affected.

Sex

Pearson syndrome has no sex predilection.

Age

Pearson syndrome is a progressive disease, and its features change with age. Neonates may be well at birth, but some neonates with Pearson syndrome have low birth weight, pallor, and anemia. Hydrops fetalis has also been reported. Anemic newborns may need transfusion.

During infancy and early childhood, failure to thrive, chronic diarrhea, and progressive hepatomegaly often occur in individuals with Pearson syndrome. These conditions are punctuated by episodic crises characterized by somnolence, vomiting, electrolytic abnormalities, lactic acidosis, and hepatic insufficiency. The lactic acidosis may become persistent with time. Typical causes of death in infants and young children with Pearson syndrome are metabolic crisis, hepatic failure, and overwhelming sepsis due to neutropenia.

Some patients survive infancy and early childhood and spontaneously recover from the hematologic dysfunction. Case reports document a shift in the phenotype of these individuals to a predominantly myopathic or encephalopathic condition. For example, some patients who survive early childhood may develop KSS or Leigh syndrome, whereas others may be neurologically healthy.

History

·  Although the history is nonspecific, the astute clinician recognizes the need for further evaluation.

·  Parents and/or caregivers may notice that the infant has been pale since birth, perhaps increasingly so; this finding indicates refractory anemia.

·  Chronic diarrhea and fatty stools may be reported and indicate pancreatic exocrine deficiency.

·  Inquire about previous illnesses or hospitalizations. Episodes of anorexia, vomiting, fever, and lethargy can occur. Associated dehydration, electrolytic abnormalities, lactic acidosis, and hepatic dysfunction may occur.

·  Inquire about weight and obtain a growth chart. The birth weight may have been low, and the infant may fail to gain weight.

·  A dietary history is important because deficiencies of copper, riboflavin, and phenylalanine may cause anemia with vacuolization of hematopoietic precursors, similar to changes observed in Pearson syndrome.

·  Obtain a history of exposure to drugs. Certain drugs can damage the bone marrow. For example, chloramphenicol can cause sideroblastic changes and vacuolization of hematopoietic precursors, similar to its effects in individuals with Pearson syndrome.

·  Obtain a family history.

o Some anemias and syndromes of bone marrow failure, such as X-linked sideroblastic anemia and Diamond-Blackfan anemia, affect families. A good family history can alert the clinician to these possible diagnoses.

o Although mitochondropathies can be inherited maternally, Pearson syndrome appears to be sporadic.

Physical

·  No pathognomonic physical characteristics are observed.

·  Anemia causes pallor.

·  The patient’s weight may be low for his or her age, and some patients are cachectic.

·  Hepatomegaly, often progressive, occurs in patients with hepatic involvement.

·  Patchy erythema and photosensitivity are also reported.

·  Examine the patient for anomalies associated with other syndromes of bone marrow failure that present in the neonate or infant. For example, anomalies of the radii and thumb suggest Fanconi anemia, Diamond-Blackfan anemia, or the thrombocytopenia-absent radii syndrome.

Causes

Several types of abnormalities of mitochondrial DNA cause Pearson syndrome.

Other Problems to Be Considered

Shwachman-Diamond syndrome is the combination of pancreatic exocrine insufficiency and neutropenia. Epiphyseal and metaphyseal dysostosis also occur in Shwachman-Diamond syndrome. Patients with Pearson syndrome may be neutropenic, but severe anemia is most characteristic.

Hereditary sideroblastic anemia lacks the characteristic vacuolization of marrow precursors, and no concomitant pancreatic insufficiency occurs. Sideroblastic anemia may respond to pyridoxine or pyridoxal phosphate.

Copper deficiency can be differentiated from Pearson syndrome on the basis of a low serum copper concentration and improvement with supplemental administration of copper.

Fanconi anemia is a congenital bone marrow failure syndrome that can be distinguished from Pearson syndrome by performing physical examination, by examining the bone marrow, and by testing for chromosomal fragility. Individuals with Fanconi anemia may have short stature, hyperpigmentation, anomalies of the thumb and radius, and other congenital abnormalities. No vacuolization of hematopoietic precursors occurs in Fanconi anemia, and chromosomes from patients with Fanconi anemia develop breaks when incubated with diepoxybutane. The cytopenias of Fanconi anemia often improve with androgen therapy.

Diamond-Blackfan anemia is congenital pure red cell aplasia characterized by isolated, severe, macrocytic anemia and often bony abnormalities of the thumbs and radii. Serum adenosine deaminase levels are usually increased in Diamond-Blackfan anemia, and no pancreatic insufficiency is observed. Many cases of Diamond-Blackfan anemia respond to glucocorticoid therapy.

Laboratory Studies

·  CBC count determination with differential and reticulocyte count

o Patients with Pearson syndrome have macrocytic anemia.

o The reticulocyte count is inappropriately low.

o Some patients also have neutropenia, thrombocytopenia, or both.

·  Test of pancreatic exocrine function

o Document evidence of pancreatic exocrine dysfunction.

o Various direct and indirect tests are available, including the following:

§ Measurement of secretory capacity induced by exogenous hormones, a test meal, or a duodenal stimulant

§ Stool microscopy and analysis of fecal fat and nitrogen

§ Measurement of serum pancreatic isoamylase, trypsinogen, and lipase concentrations

·  Measurement of serum lactic acid

o Patients may have lactic acidemia, though it may be intermittent.

o The ratio of lactate to pyruvate may be increased.

·  Urinalysis

o Complex organic aciduria, including 3-methylglutaconic aciduria, is reported.

o Some patients have proximal renal tubular dysfunction that causes urinary wasting of amino acids, glucose, bicarbonate, phosphate, citrate, and urate.

·  Hepatic study

o Hepatic transaminase values may be increased in patients with hepatic involvement.

o Bilirubin levels may be increased, and albumin concentrations and coagulation values (eg, prothrombin time) may reflect a defect in synthetic function.

·  Endocrinologic study: Some patients have hypothyroid, hypoparathyroid, and a deficiency in growth hormone.

·  Analysis of mitochondrial DNA

o The causative deletions of mitochondrial DNA can be demonstrated with molecular genetic analysis. Because of heteroplasmy, not all tissues contain abundant amounts of mutant mitochondrial DNA.

o Bone marrow cells are appropriate for sampling. Peripheral blood cells are also appropriate for mitochondrial DNA analysis. However, because of heteroplasmy, mutant DNA may not always be found. If Pearson syndrome is suspected despite normal findings in other tissues, analysis of bone marrow is prudent.

Imaging Studies

·  No specific imaging studies are needed to diagnose Pearson syndrome.

·  MRI of the brain may be performed to further investigate a phenotypic shift to a predominantly encephalopathic or myopathic condition, which may occur in older individuals with Pearson syndrome.

Procedures

Bone marrow aspiration and biopsy are necessary to obtain bone marrow for histologic analysis. Characteristic histologic findings of Pearson syndrome can be observed, and other causes of pancytopenia can be excluded.

Histologic Findings

The number of erythroid precursors in the bone marrow is normal or increased, and a characteristic vacuolization of hematopoietic precursors occurs (see Images 1-2).

Characteristic vacuolization of a hematopoietic precursor in the bone marrow. (Light microscopy; 100x; Wright-Giemsa stain)

 

Electron photomicrograph of a hematopoietic precursor (normoblast) with vacuolization. (Transmission electron microscopy; original 10,000x)

 

An increased number of sideroblasts with ringed sideroblasts may be observed on iron staining (see Image 3).

Ringed sideroblast in the bone marrow (iron stain). The dark structures that form a ring around the nucleus are hemosiderin-laden mitochondria. (Light microscopy; 100x; iron stain)

Medical Care

No specific therapy is available for individuals with Pearson syndrome or other mitochondrial cytopathies. Attentive care and awareness of possible complications may prevent death and minimize morbidity.

·  Patients with Pearson syndrome ofteeed transfusions to manage anemia, and many are dependent on transfusions. Some hematologists use erythropoietin to decrease the frequency of transfusions.

·  Pancreatic enzyme replacement is needed for patients with malabsorption due to exocrine pancreatic insufficiency. Supplementation with fat-soluble vitamins may also be needed.

·  Evaluate fever promptly. Parenteral antimicrobials, after the blood is cultured, are required for patients who are neutropenic. Splenic atrophy may increase the risk of bacterial sepsis. Granulocyte colony-stimulating factor (G-CSF) has been used in some patients to prevent or treat severe neutropenia.

·  Manage intermittent metabolic crises with hydration, correction of electrolyte abnormalities, correction of acidosis, and a search for underlying causes (eg, infection). Seek evidence of concomitant hepatic failure. Chronic bicarbonate supplementation and dichloroacetic acid have been used to treat persistent metabolic acidosis.

·  Patients may have hypothyroidism, hypoparathyroidism, diabetes mellitus, or growth hormone deficiency. These conditions need appropriate treatment.

·  Stem cell transplantation has been reported in only one individual with Pearson syndrome.¹ Pearson syndrome is a multisystem disorder, thus, transplantation can only correct the hematologic manifestations of the disorder and cannot correct the dysfunction of other systems. Transplantation may be associated with unique or greater than expected toxicities as well.

Surgical Care

·  No specific surgical management is needed for patients with Pearson syndrome.

·  Some patients may benefit from an indwelling venous catheter to facilitate frequent transfusions or infusions.

Consultations

Consultation and collaboration with an expert in metabolism and genetics are prudent.

Diet

No dietary restrictions or modifications are required.

Activity

No specific restrictions to activity are required. Patients with neuromuscular manifestations may require appropriate support.

No specific therapy is available for individuals with Pearson syndrome or other mitochondrial cytopathies. Attentive care and awareness of possible complications may prevent death and minimize morbidity. Anecdotal reports describe the use of long-term bicarbonate supplementation and dichloroacetic acid to manage persistent metabolic acidosis.

MULTIFACTORIAL INHERITANCE

IMPORTANCE

 

Multifactorial inheritance is responsible for the greatest number of individuals that will need special care or hospitalization because of genetic diseases. Up to 10% of newborn children will express a multifactorial disease at some time in their life. Atopic reactions, diabetes, cancer, spina bifida/anencephaly, pyloric stenosis, cleft lip, cleft palate, congenital hip dysplasia, club foot, and a host of other diseases all result from multifactorial inheritance. Some of these diseases occur more frequently in males. Others occur more frequently in females. Environmental factors as well as genetic factors are involved.

REGRESSION TO THE MEAN

Multifactorial inheritance was first studied by Galton, a close relative of Darwin and a contemporary of Mendel. Galton established the principle of what he termed “regression to mediocrity.” Mendel studied discontinuous characters, green peas vs. yellow peas, tall vs. dwarf, etc. There was no overlap of phenotype in Mendel’s studies. Characters fit into one of two classes. There was no blending in the heterozygote. On the other hand, Galton studied the inheritance of continuous characters, height in humans, intelligence in humans, etc. Galtooticed that extremely tall fathers tended to have sons shorter than themselves, and extremely short fathers tended to have sons taller than themselves. “Tallness” or “shortness” didn’t breed true like they did in Mendel’s pea experiments. The offspring seemed to regress to the median, or “mediocrity.” Figure 12 shows the correlation between the father’s height and the height of the son.

 

 

 

Figure 12. A representation of GaltonХs studies on the inheritance of height. If the sonХs height were determined only by the fatherХs height, the correlation should be that of the solid line. The dashed line is what is observed. Galton called this “regression to mediocrity.”

 

If the son’s height were completely determined by the father’s height, the correlation would be as shown by the solid blue line. What is observed is shown by the dashed red line. The height of the father and the average height of the son are related, but the average height of the son always regresses toward the mean. That is understandable if there is no dominance. The son only gets half of his father’s genes; the other half comes from his mother.

 

When comparing height differences between men and women, women are, on average, 3 inches shorter. A woman with a certaiumber of “tall” genes will be, on average, 3 inches shorter than a man with the same number. When that difference is taken into account, there is no selective bias in matings for tallness in human populations. It is true than men tend to marry women who are shorter than themselves, but that is a phenotypic difference, not a genotypic difference. Since the wives of taller than average men tend to represent the general population of women, they will not have, on the average, as many “tall” genes to pass on to their offspring as their husbands. Hence, the son will receive half of the father’s “tall” genes, on average, and half of the mother’s “tall” genes, on average, but his total genes for “tallness,” on average, will be less than his father’s. Shorter than average males have fewer “tall” genes than average, but they are still as tall as an average female, even though the average female has more “tall” genes. Their sons, on average, will be taller than their fathers because their mothers have, on average, more “tall” genes to give to their sons than their husbands have. On average, the son will have more “tall” genes than his father.

What holds true for height also holds true for other quantitative traits, such as intelligence. This is what worried Galton. He was a very intelligent member of British aristocracy who was interested in genetics as a way to maintain intelligence in his family. He was really the founder of the eugenics movement. His findings must have been very discouraging for him.

 

POLYGENIC INHERITANCE

For many years the argument raged between the “Mendelians” and the “Galtonians” as to which of the two paradigms was the correct one for human inheritance. There was no question that Mendelian inheritance was correct for some diseases, but these were rare, affecting only a small portion of the population. They were considered trivial by the Galtonians. On the other hand, the inheritance of quantitative traits could not be used to predict outcomes, only average estimates measured in large population studies. Mendelians considered the study of quantitative traits to be trivial because they had no predictive value. R. A. Fisher resolved the dispute by showing that the inheritance of quantitative traits can be reduced to Mendelian inheritance at many loci. Fisher’s argument went as follows:

Consider the following: One locus for height, with three alleles. Allele h2 adds 2 inches to the average 68-inch height. Allele h0 neither adds nor subtracts from the average height of 68 inches. And allele h- subtracts 2 inches from the average height. Suppose h0 is twice as frequent as either h2 or h-.

When this is expressed in tabular form, it looks like the histogram in Figure 13.

 

Figure 13. The distribution of height in a population if it were determined by one locus with three alleles as described in the text.

 

If a second locus, called the tall locus, or t, is also involved in height, with three alleles as above, one adding two inches, one neither adding nor subtracting from the phenotype, and one subtracting 2 inches, with the neutral allele occurring twice as frequently as the either of the others, the histogram becomes that of Figure 14.

 

Figure 14. The distribution of height in a population if were determined by two loci, each with three alleles as described in the text.

THE MULTIFACTORIAL MODEL

 

As more loci are included, this binomial distribution quickly approaches the Gaussian distribution, or the bell-shaped normal curve, observed with human quantitative traits. Three loci, each with three alleles, are enough to produce population frequencies indistinguishable from a normal curve. The multifactorial model is then:

Several, but not an unlimited number, loci are involved in the expression of the trait.

There is no dominance or recessivity at each of these loci.

The loci act in concert in an additive fashion, each adding or detracting a small amount from the phenotype.

The environment interacts with the genotype to produce the final phenotype.

 

As an example of 4. above, women are, on average, three inches shorter than men with the same genome. Environmental factors (hormones) affect the final phenotype.

 

Not all human traits that show a continuous distribution in the population are multifactorial traits. Any bimodal distribution is not controlled by multifactorial expression. It is more likely to be under the control of a single dominant/recessive gene with modifying environmental factors. Multifactorial traits all show a unimodal bell-shaped distribution.

CONCORDANCE

 

Twin studies, although limited by complicating factors, provide the best source for separating genetic contributions to the trait being studied from environmental influences. Monozygous (identical) twins have the same genome, but not the exact environmental factors, especially if they were raised apart. The concordance rate in monozygotic twins can be compared to the concordance rate in dizygotic (fraternal) twins to estimate the genetic component (heritability) of the trait. If the trait is truly 100% genetic, as it is for total fingerprint ridge count in humans, monozygotic twins will be 100% concordant while dizygotic twins, having, on average, only half their genes in common, will have a lower concordance rate. If the trait under study is 100% environmental, monozygotic twins and dizygotic twins will have the same concordance rate. The concordance rate for a disease is calculated as follows:

 

Concordance Rate = [Both Affected / (One Affected + Both Affected)] x 100

 

For quantitative traits, means and variances have to be substituted and the calculations are beyond the scope of this introductory course.

THRESHOLD MODEL OF DISEASE

 

If multifactorial traits are quantitative traits with continuous distribution, how can they control diseases, such as cleft lip or spina bifida? One either has the disease or doesn’t. There is no intermediate. I’m glad you asked that question. Multifactorial diseases are best explained by the threshold model shown in Figure 15

 

Figure 15. The threshold model for multifactorial traits. Below the threshold the trait is not expressed. Individuals above the threshold have the disease.

 

As the number of multifactorial genes for the trait increases, the liability for the disease increases. When it reaches a threshold, the liability is so great that abnormality, what we call disease, results. For example, consider the development of the cleft palate. Early in embryonic development the palatal arches are in a vertical position. Through embryonic and fetal development the head grows larger, making the arches farther apart, the tongue increases in size, making it more difficult to move, and the arches themselves are growing and turning horizontal. There is a critical stage in development by which the two arches must meet and fuse. Head growth, tongue growth, and palatal arch growth are all subject to many genetic and environmental factors. If the two arches start to grow in time, grow at the proper rate, and begin to move soon enough to the horizontal they will meet and fuse in spite of head size and tongue growth. The result is no cleft palate. They may fuse well ahead of the critical developmental stage or just barely make it in time, we have no way of telling. However, if they don’t meet by the critical stage a cleft palate results. If they are close together at the critical stage, a small cleft will result, perhaps only a bifurcated uvula. If they are far apart, a more severe cleft will result. We call that critical difference in liability the threshold. Beyond the threshold, disease results. Below the threshold, normal development is observed. But the underlying liability is distributed as the normal curve shown in Figure 15.

 

DEGREE OF RELATIONSHIP AND GENES IN COMMON

 

Since one is not following a single locus with dominance or recessivity but is following several loci that act in concert, counseling for multifactorial inheritance diseases requires a different approach from that taken for Mendelian inheritance diseases. One has to calculate the number of genes in common. The easiest way to do that is to change the way we construct pedigrees. Instead of the familiar sibship method we use the pathway to common ancestor method. It is shown in Figure 16.

 

Figure 16. Conversion of a standard pedigree to a path coefficient pedigree for determining the fraction of genes in common.

 

In Figure 16, Pedigree A represents the standard method of pedigree construction. Pedigree B represents the pathway system of pedigree construction. It is much easier to see how genes flow from generation to generation in Pedigree B. In Figure 16, II-2 and II-3 are brother and sister. They have two common ancestors, I-1 and I-2. To determine the fraction of genes II-2 and II-3 have in common one simply counts all of the pathways and their connecting lines through the common ancestors. There is one line from II-2 to I-1, and a line from I-1 to II-3. That is one pathway with two lines of descent. There is another line from II-2 to I-2, and a line from I-2 to II-3. That is a second pathway with two lines of descent. These are the only pathways from II-2 to II-3. The fraction 1/2 is then raised to the power of the number of lines of descent and summed for each possible pathway, (1/2)2 for the pathway through I-1, and (1/2)2 for the pathway through I-2, making a total of 1/2. Brothers and sisters have, on average, 1/2 of their genes in common.

 

A parent and offspring, say I-1 and II-2 also have 1/2 of their genes in common. There is only one pathway between them and only one line in that pathway, (1/2)1.

 

Other relationships follow in the same manner. In Figure 16, III-1 and III-3 are first cousins. There are two pathways connecting the two individuals, one through I-1 and the other through I-2, each with four lines. Their fraction of genes in common is then (1/2)4 + (1/2)4 or 1/8. First cousins have 1/8 of their genes in common. A grandparent and grandchild have 1/4 of their genes in common. There is a single pathway with two lines of descent. III-1 and IV-1 are first cousins once removed. Again there are two pathways, one through I-1 and the other through I-2, each with 5 lines, (1/2)5 + (1/2)5 or 1/16 of their genes in common.

 

The degree of relationship is often used rather than the fraction of genes in common. The degree of relationship is simply the power to which (1/2) is raised to reach the fraction of genes in common. First degree relatives have (1/2) of their genes in common. Second degree relatives have 1/4, (1/2)2, of their genes in common, etc.

 

Figure 17. Method of calculating the recurrence risk of a multifactorial trait to first degree relatives.

 

If one returns to the normal curve for liability shown in Figure 15, one caow see where various relatives of affected lie in relationship to the threshold. Figure 17 shows these calculations for first degree relatives. The mean of affected can be calculated by dividing the affected rate by 2 and plotting that area under the normal curve. If a multifactorial disease affects 1/2000 offspring of normal x normal matings, then the mean of affected is an area of 1/4000. The number of standard deviations this area is from the mean can be found in statistical tables. Since first degree relatives have 1/2 their genes in common with their affected relative, first degree relatives of affected will be 1/2 of the way between the mean for affected and the population mean. This can also be calculated. A new normal curve with the same variance is then plotted using the mean for first degree relatives. The threshold does not move, so the overlap of the threshold will give the probability of recurrence to first degree relatives of affected. This is shown in Figure 18. Don’t worry, you won’t be asked to do this on an examination. This was done solely for the purpose of demonstrating the method for calculating the recurrence risk to relatives of affected. In practice you will simply look up the disease in the proper atlas and find the recurrence risks listed.

 

Figure 18. Recurrence risk to first degree relatives of affected individuals.

TWO THRESHOLD DISEASES

In many multifactorial diseases the two sexes have different probabilities of being affected. For example, pyloric stenosis occurs in about 1/200 newborn males but only in about 1/1000 newborn females. This means that there is a double threshold, one for females and one for males, with the female threshold farther from the mean than that for the male. However, since it takes more deleterious genes to create an affected female, she has more genes to pass on to the next generation. Her male offspring are at a relative high risk of being affected when compared to the population risk.

SEVERITY OF DISEASE AND RECURRENCE RISK

Unlike Mendelian traits with variable expressivity, where the recurrence risk is the same no matter how severely the individual is affected, multifactorial traits have a higher recurrence risk if the relative is more severely affected. In multifactorial traits, the more severely affected the individual, the more genes he/she has to transmit, and the higher the recurrence risk.

MULTIPLE AFFECTED OFFSPRING AND RECURRENCE RISK

Another difference is the presence of multiple affected individuals within a sibship. In Mendelian traits the number of affected in a family did not change the recurrence risks. But multiple affected children does change the recurrence risk for multifactorial traits. The presence of one affected child means the parents probably are midway between the mean for affected and the mean of the normal population, but the presence of a second affected child means they probably are closer to the threshold, and hence, have a higher recurrence risk should they choose to have another child.

CONSANGUINITY

Consanguinity also increases the probability of an affected child for a multifactorial trait, but only slightly when compared to rare autosomal recessive diseases. First cousin matings may increase the risk for two normal individuals to have a child with a multifactorial disease by about two fold when compared to the risk for unrelated individuals.

 

HALLMARKS OF MULTIFACTORIAL INHERITANCE

In summary, the hallmarks for multifactorial inheritance are:

Most affected children have normal parents. This is true of diseases and quantitative traits. Most geniuses come from normal parents, most mentally challenged come from normal parents.

Recurrence risk increases with the number of affected children in a family.

Recurrence risk increases with severity of the defect. A more severely affected parent is more likely to produce an affected child.

Consanguinity slightly increases the risk for an affected child.

Risk of affected relatives falls off very quickly with the degree of relationship. Contrast this with autosomal dominant inheritance with invomplete penetrance, where the recurrence risk falls off proportionately with the degree of relationship.

If the two sexes have a different probability of being affected, the least likely sex, if affected, is the most likely sex to produce an affected offspring.

 

Genetic Counseling

 

Genetic counseling is the process of providing individuals and families with information on the nature, inheritance, and implications of genetic disorders to help them make informed medical and personal decisions. The following section deals with genetic risk assessment and the use of family history and genetic testing to clarify genetic status for family members. This section is not meant to address all personal, cultural, or ethical issues that individuals may face or to substitute for consultation with a genetics professional. To find a genetics or prenatal diagnosis clinic, see the GeneTests Clinic Directory.

 

When a child is born with multiple congenital anomalies or a family is diagnosed with a genetic disorder, talking with the family is not easy. Giving bad news is always difficult and the information is often somewhat technical. However, it is important to provide the family with as much information as possible in order for them to make informed decisions. Genetic counseling has been defined as “an educational process that seeks to assist affected and/or at risk individuals to understand the nature of a genetic disorder, its transmission and the options available to them in management and family planning.”

In recent years the task of providing information about genetic diseases has been done with a team approach using highly trained medical geneticists and genetic counselors, but this information can also be provided by a family physician, a pediatrician, or a nurse. Genetic counseling must be done based on an understanding of genetic principles, the ability to recognize and diagnose genetic diseases and rare syndromes, and knowledge of the natural history of the disorder and its recurrence risk. Awareness of prenatal diagnosis and screening programs available in a particular region and access to information about new advances in genetic disorders and techniques are necessary.

TALKING TO FAMILIES. The type of information provided to a family depends on the urgency of the situation, the need to make decisions, or the need to collect additional information. However, in simple terms there are three general situations in which genetic  counseling becomes particularly important.

The first situation is the prenatal diagnosis of a congenital anomaly or genetic disease. This is a very difficult situation, and the need for information is urgent because a family must often decide whether to continue or terminate a pregnancy. The second type of situation occurs when a child is born with a congenital anomaly or genetic disease. This also requires urgent information and decisions must be made immediately with regard to how much support should be provided for the child and whether certain types of therapy should be attempted. The third situation arises later in life when (1) a diagnosis with a genetic implication is made, (2) a couple is planning a family and there is a family history of the problem (e.g,. a couple in which one carries a translocation or is a carrier of cystic fibrosis), or (3) when an adolescent or young adult has a family history of an adult-onset genetic  disorder  (e.g., Huntington disease or breast cancer).  It is ofteecessary to have several meetings with a   family,   because all   of   their questions and concerns cannot be addressed at one time.

GENETIC COUNSELING. Providing accurate information to families requires

taking a careful family history and constructing a pedigree that lists the patients’ relatives (including abortions, stillbirths,  and deceased individuals)  with their sex,  age,  and state of  health;

o       gathering information from hospital records about the affected individual (and in some cases, other family members);

o       documenting the prenatal, pregnancy and delivery history;

o       reviewing the available information concerning the disorder;

o       careful physical examination of the affected individual (with photographs and measurements) and of apparently unaffected individuals;

o       establishing or confirming the diagnosis  by  the  diagnostic   tests available;  

o       giving the family information about support groups;

o       providing new information to the family as it becomes available.

In order to provide optimal benefits, the counseling session must include certain information.

Knowledge of the Diagnosis of the Particular Condition. Although not always possible to make an exact diagnosis, having as accurate a diagnosis as possible is important. Estimates of recurrence risk for various family members depend on an accurate diagno­sis. When a specific diagnosis cannot be made (as in many cases of multiple congenital anomalies), the various differential diagnoses should be discussed with the family and empirical information provided. If specific diagnostic tests are available, they should be discussed.

Natural History of the Condition. It is very important to discuss the natural history of the specific genetic disorder(s) in the family. Affected individuals and their families will have questions regarding the prognosis and potential therapy that can only be answered with knowledge of the natural history. If there are other possible differential diagnoses, their natural history may also be discussed. If the disorder is associated with a spectrum of clinical outcomes or complications, the worst and best scenario, as well as treatment and referral to the appropriate specialist, should be addressed.

Genetic Aspects of the Condition and Recurrence Risk. This is important information for the family because family members need to be aware of their reproductive choices. The genetics of the disorder can be explained with visual aids (i.e., figures of chromosomes, etc.). It is important to provide accurate occurrence and recurrence risks for various members of the family, including unaffected individuals, cousins, aunts, and so forth. In cases in which a definite diagnosis cannot be made, it will be necessary to use empirical recurrence risks. Counseling should give the individuals the necessary information to understand various options and to make their own informed decisions regarding pregnancies, abortion, artificial insemination, prenatal diagnosis, screening, carrier detection, and termination of pregnancy. In order to complete the educational process, it may be necessary to have more than one counseling session.

Prenatal Diagnosis and Prevention. There are many different methods of prenatal diagnosis available depending on the specific ge­netic disorder. The use of ultrasound allows prenatal diagnosis of anatomic abnormalities such as neural tube defects. Amniocentesis and chorionic villus samples (CVS) are used to obtain fetal tissue for analysis of chromosomal abnormalities, biochemical disorders, and DNA studies. Maternal blood or serum samples are used for some types of screening.

Therapies and Referral. There are a number of genetic disorders that require the care of a specialist. For example, individuals with Turner syndrome usually need to be evaluated by an endocrinologist. Prevention of known complications is a priority. The psychological adjustment of the family may require specific intervention.

Support Groups. Over the last few years a large number of law support groups have been formed to provide information and fund research on specific genetic and nongenetic conditions. An important part of genetic counseling is to give information about these groups to individuals and to be able to suggest a contact person for the families.

Follow-up. Families should be encouraged to continue to ask questions and keep up with new information about the specific disorder. New developments often influence the diagnosis and therapy of specific genetic disorders. Law groups are a good source of new information.

 

DNA diagnostics.

R. Daniel Gietz Ph.D

 

Learning Objectives.

After this lecture the student should be able to;

1.      Understand some the basic principles underlying common DNA Diagnostic tests.

2.      Understand the differences and advantages and limitations of each DNA Diagnostic test.

3.      Appreciate the issues related to DNA testing such as presymptomatic testing, insurability and testing of children.

Population Screening for Genetic Disease.

This type of screening is an important component of routine health care.  Examples; pap test for the recognition of cervical dysplasia and screening for hypercholestrolemia.   It is the “presumptive identification of an unrecognized disease or defect by the application of tests, examinations or other procedures which can be applied rapidly to sort out apparently well persons who probably have a disease from whose who probably do not” (Mausner and Bahn, 1974 Epidemiology: An introductory Text W.B. Saunders, Philidelphia).

Genetic screening is defined as “search in the population for persons possessing certain genotypes that: (1) are already associated with disease or predisposition to disease, or  (2) may lead to disease in their descendants” (National Academy of Sciences , 1975, Genetic Screening; Programs, Principals and Research).  Examples of genetic screening are; Newborn screening for inherited metabolic disorders such as TaySachs.

Principles of Screening

1. Disease Characteristics, The disease should be serious and relatively common.  There should be a cost/benefit justification.  The natural history of the disease should be clearly understood.  There should be acceptable and effective treatment or in the case of genetic conditions prenatal diagnosis should be available.

2. Test Characteristics, The test should be acceptable, easy to perform, relatively inexpensive and be reliable and valid.

3. System Characteristics, The resources for diagnosis and treatment must be accessible and a strategy for communicating the result efficiently and effectively must be in place. 

Test Validity

 There are two components that speak to a tests validity, its sensitivity and its specificity. 

Sensitivity :  the ability to correctly identify those with the disease (true positive).  It is measured as the proportion of the affected individuals in whom the test is positive. 

Specificity:  The ability of to correctly identify those without the disease (true negative)  It is measured as the proportion of the unaffected in whom the test is negative. 

Tests are rarely 100% sensitive nor 100% specific. 

 

 

1. New born Screening is an effective public health strategy for treatable disorders.

            PKU, Galactosemia, sickle cell anemia, Duchene muscular dystrophy 

See Table1 below

 

Table 1 Characteristics of Selected Newborn Screening Programs
Disease	Inheritance	Prevalence	Screening test	Cost	Treatment
PKU	Autosomal recessive	1/10,000 –1/15,000	Guthrie Test	$1.25	Dietary restriction of Phe
Galactosemia	Autosomal recessive	1/50,000 – 1/1000,000	Transferase assay	$1.00	Dietary restriction of galactose
Congentital Hypothyroidism	Sporatic	1/5000	Measurement of T4 or TSH	$1.50	Hormone replacement
Sickle Cell disease	Autosomal recessive	1/400 – 1/600 African Americans	Isoelectric Focusing or DNA diagnostic	$1.50	Prophylactic penicillin

 

 

2. Heterzygote Screening is the testing of a target population to identify unaffected carriers of a disease gene.  The carriers are given information about risks and reproductive options.  Example Tay Sachs disease, Cystic Fibrosis.  See Table 2

 

Table 2 Examples of Heterozygote screening programs in specific ethnic groups
Disease	Ethnic group	Carrier frequency	At risks couple frequency	Disease incidence iewborns
Sickle cell disease	African American	1/12	1/150	1/600
Tay-Sachs disease	Ashkenazi Jews	1/30	1/900	1/3600
B-Thalassemia	Greeks, Italians	1/30	1/900	1/3600
A-Thalassemia	South east Asians, Chinese	1/25	1/625	1/2500
Cystic Fibrosis	Northern Europeans	1/25	1/625	1/2500

 

 

3. Presymptomatic Diagnosis is the testing of individuals who may have inherited a disease causing gene before they develop symptoms.

 

 

Basic:

1.           Medical Genetics + Student Consult, 4th Edition. Lynn B. Jorde, John C. Carey, MPH and Michael J. Bamshad,  2010, p. 368. ISBN: 978-032-305-373-0

2.           Essential Medical Genetics, 6 edition. Edward S. Tobias, Michael Connor, Malcolm Ferguson Smith. Published by Wiley-Blackwell, 2011, p. 344. ISBN: 978-140-516-974-5 

3.           Emery’s Elements of Medical Genetics + Student Consult, 14th Edition Peter D Turnpenny,  2012 p. 464. ISBN: 978-070-204-043-6   

4.           Genes, Chromosomes, and Disease: From Simple Traits, to Complex Traits, to Personalized Medicine. Nicholas Wright Gillham. Published by FT Press Science, 2011. p. 352.

5.            Management of Genetic Syndromes. Suzanne B. Cassidy, Judith E. Allanson; 3 edition.  Published by Wiley-Blackwell. 2010 p. 984 ISBN: 978-047-019-141-5.

6.           Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment (Milunsky, Genetic Disorders and the Fetus) 6 edition. Aubrey Milunsky and Jeff Milunsky. Published by Wiley-Blackwell, 2010 p. 1184 ISBN: 978-140-519-087-9

7.           Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. 1st edition. Buckingham L, Flaws ML. F.A. Davis. 2007.

8.           Passarge E. Color Atlas of Genetics – Thieme, 2007 Р. 497

 

Additional:

1.           Atlas of Inherited Metabolic Diseases 3 edition William L Nyhan  Bruce A Barshop, Aida I Al-Aqeel. Published by CRC. Press 2011,  p 888. ISBN: 978-144-411-225-2.

2.           Inborn Metabolic Diseases: Diagnosis and Treatment Jean-Marie Saudubray, Georges van den Berghe, John H. Walter. 5th ed. Published by Springer, 2012, p. 684. ISBN: 978-364-215-719-6.

3.           Chromosome Abnormalities and Genetic Counseling (Oxford Monographs on Medical Genetics). 4 edition R. J. M. Gardner, Grant R Sutherland and Lisa G. Shaffer. Published by Oxford University Press, USA; 2011 p.648. ISBN: 978-019-537-533-6.

4.           Mitochondrial Medicine: Mitochondrial Metabolism, Diseases, Diagnosis and Therapy Editored by Anna Gvozdjáková. Published by Soringer, 2008, p. 409 ISBN 978-1-4020-6713-6 

5.           Mitochondrial DNA, Mitochondria, Disease and Stem Cells (Stem Cell Biology and Regenerative Medicine) Editor Justin C. St. John. Published by Humana Press, 2012 p.199. ISBN: 978-162-703-100-4.

6.           Genetic Counseling Practice: Advanced Concepts and Skills. Bonnie S. LeRoy, Patricia M. Veach, Dianne M. Bartels. Published by Wiley-Blackwell, 2010, p. 415. ISBN: 978-047-018-355-7

7.           A Guide to Genetic Counseling 2 edition; Wendy R. Uhlmann, Jane L. Schuette, Beverly Yashar.  Published by Wiley-Blackwell, 2010, p. 644. ISBN: 978-047-017-965-9

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