Lecture
The subject and the main tasks of Medical Genetics. Role of heredity in human pathology.
Genetics is defined in dictionaries as the science that deals with heredity and variation in organisms, including the genetic features and constitution of a single organism, species, or group, and with the mechanisms by which they are effected (Encyclopaedia Britannica 15th edition, 1995; Collins English Dictionary, 5th edition2001).
New investigative methods and observations, specially during the last 50 years, have moved genetics into the mainstream of biology and medicine. Genetics is relevant to virtually all fields of medicine and biological disciplines, anthropology, biochemistry, physiology, psychology, ecology, and other fields of the sciences. As both a theoretical and an experimental science, it has broad practical applications in understanding and control of genetic diseases and in agriculture. Knowledge of basic genetic principles and their medical application is an essential part of medical education today. The determination of the nearly complete sequence of the building blocks encoding the genetic information of man in 2004 marked an unprecedented scientific milestone in biology.
The Human Genome Project, an internationalorganization of several countries, reported this major achievement just 50 years after the structure of DNA, the molecule that encodes genetic information, was elucidated (IHGSC, 2004). Although much work remains before we know how the molecules of life interact and produce living organisms, through genetics we now have a good foundation for understanding the living world from a biological perspective.
Each of the approximately ten trillion (1013) cells of an adult human contains a program with life-sustaining information in its nucleus (except red blood cells, which do not have a nucleus). This information is hereditary, transmitted from one cell to its descendent cells, and from one generation to the next. About 200 different types of cells carry out the complex molecular transactions required for life. Genetic information allows organisms to convert atmospheric oxygen and ingested food into energy production, it regulates the synthesis and transport of biologically important molecules, protects against unwarranted invaders, such as bacteria, fungi, and viruses by means of an elaborate immune defense system, and maintains the shape and mobility of bones, muscles, and skin.
Genetically determined functions of the sensory organs enable us to see, to hear, to taste, to feel heat, cold, and pain, to communicate by speech, to support brain function with the ability to learn from experience, and to integrate the environmental input into cognate behavior and social interaction. Reproduction and detoxification of exogenous molecules likewise are under genetic control. Yet, the human brain is endowed with the ability to take free decisions in daily life and developing plans for the future.
The living world consists of two types of cells, the smallest membrane-bound units capable of independent reproduction: prokaryotic cells without a nucleus, represented by bacteria, and eukaryotic cells with a nucleus and complex internal structures, which make up higher organisms. Genetic information is transferred from one cell to both daughter cells at each cell division and from one generation to the next through specialized cells, the germ cells, oocytes, and spermatozoa. The integrity of the genetic program must be maintained without compromise, yet it must be adaptable to long-term changes in the environment.
Errors in maintaining and transmitting genetic information occur frequently in all living systems despite the existence of complex systems for damage recognition and repair. Biological processes are mediated by biochemical reactions performed by biomolecules, called proteins. Each protein is made up of dozens to several hundreds of amino acids arranged in a linear sequence that is specific for its function. Subsequently, it assumes a specific three-dimensional structure, often in combination with other polypeptides. Only this latter feature allows biological function. Genetic information is the blueprint for producing the proteins in a given cell. Most cells do not produce all possible proteins, but a selection, depending on the type of cell. The instructions are encoded in discrete units, the genes. Each of the 20 amino acids used by living organisms recognizes a code of three specific chemical structures. These are the nucleotide bases of a large molecule, DNA (deoxyribonucleic acid).DNA
DNA is a read-only memory device of a genetic information system, called the genetic code. In contrast to the binary system of strings of ones and zeros used in computers (“bits,”which are then combined into “bytes,” which are eight binary digits long), the genetic code in the living world uses a quaternary system of four nucleotide bases with chemical names having the initial letters A, C, G, and T. The quaternary code used in living cells uses three building blocks, called a triplet codon. This genetic code is universal and is used by all living cells, including plants and viruses. A gene is a unit of genetic information.It is equivalent to a single sentence in a text. Thus, genetic information is highly analogous to a linear text and is amenable to being stored in computers.
Genes
Depending on the organizational complexity of an organism, its number of genes ranges from about
termed genomics and proteomics, respectively.
Genes are located on chromosomes. Chromosomes are individual, complex structures located in the cell nucleus, consisting of DNA and special proteins. Chromosomes come in pairs of homologous chromosomes, one derived from the mother, and one from the father. Man has 23 pairs, consisting of chromosomes 1–22 and an X and a Y chromosome in males or two X chromosomes in females. The number and size of chromosomes in different organisms vary, but the total amount of DNA and the total number of genes are the same for a particular species.
Genes are arranged in linear order along each chromosome. Each gene has a defined position, called a gene locus. In higher organisms, genes are structured into contiguous sections of coding and noncoding sequences, called exons (coding) and introns (noncoding), respectively. Genes in multicellular organisms vary with respect to size (ranging from a few thousand to over a million nucleotide base pairs), the number and size of exons, and regulatory DNA sequences. The latter determine the state of activity of a gene, called gene expression. Most genes in differentiated, specialized cells are permanently turned off. Remarkably, more than 90% of the 3 billion base pairs of DNA in higher organisms do not carry known coding information.
The linear text of information contained in the coding sequences of DNA in a gene cannot be read directly. Rather, its total sequence is first transcribed into a structurally related molecule with a corresponding sequence of codons. This molecule is called RNA (ribonucleic acid). RNA is processed by removing the noncoding sections (introns). The coding sections (exons) are spliced together into the final template, called messenger RNA (mRNA). This serves as a template to arrange the amino acids in the sequence specified by the genetic code. This process is called translation.
Genes and Evolution
In The Origin of Species, Charles Darwin wrote in 1859 at the end of chapter IX, On the Imperfection of the Geological Record: “. . .I look at the natural geological record, as a history of the world imperfectly kept, and written in a changing dialect; of this history we possess the last volume alone, relating only to two or three countries. Of this volume, only here and there a short chapter has been preserved; and of each page, only here and there a fewlines.” Advances in genetics and new findings of hominid remains have provided new insights into the process of evolution. Genes with comparable functions in different organisms share structural features. Occasionally they are nearly identical. This is the result of evolution. Living organisms are related to each other by their origin from a common ancestor. Cellular life was established about 3.5 billion years ago when land masses first appeared. Genes required for fundamental functions are similar or almost identical across a,wide variety of organisms, e.g., in bacteria, yeast, insects, worms, vertebrates, mammals, and even plants. They control vital functions such as the cell cycle, DNA repair, or in embryonic development and differentiation. Similar or identical genes present in different organisms are referred to as conserved in evolution.
Genes evolve within the context of the genome of which they are a part. Evolution does not proceed by accumulation of mutations. Most mutations are detrimental to function and usually do not improve an organism’s chance of surviving. Rather, during the course of evolution existing genes are duplicated or parts of genes reshuffled and brought together in a new combination. The duplication event can involve an entire genome, a whole chromosome or a part of it, or a single gene or group of genes. All these events have been documented in the evolution of vertebrates. The human genome contains multiple sites thatwere duplicated during evolution .
Humans, Homo sapiens, are the only living species within the family of Hominidae. All data
available are consistent with the assumption that today’s humans originated in Africa about 100000 to 300000 years ago, spread out over the earth, and populated all continents. Owing to regional adaptation to climatic and other conditions, and favored by geographic isolation, different ethnic groups evolved. Human populations living in different geographic regions differ in the color of the skin, eyes, and hair. This is often mistakenly used to define human races.
However, genetic data do not support the existence of human races. Genetic differences exist mainly between individuals regardless of their ethnic origin. In a study of DNA variation from 12 populations living on five continents of the world, 93–95% of differences were between individuals; only 3–5%were between the populations. Observable differences are literally superficial and do not form a genetic basis for distinguishing races.
Genetically, Homo sapiens is one rather homogeneous species of recent origin. As a result of evolutionary history, humans are well adapted to live peacefully in relatively small groups with a similar cultural and linguistic history. However, humans have not yet adapted to global conditions. They tend to react with hostility to groups with a different cultural background in spite of negligible genetic differences.
Changes in Genes: Mutations
In 1901, H. De Vries recognized that genes can change the contents of their information. For this new observation, he introduced the term mutation. The systematic analysis of mutations contributed greatly to the developing science of genetics. In 1927, H. J. Muller determined the spontaneous mutation rate in Drosophila and demonstrated that mutations can be induced by roentgen rays. C. Auerbach and J. M. Robson in 1941 and, independently, F. Oehlkers in 1943 observed that certain chemical substances also could induce mutations. However, it remained unclear what a mutation actually was, since the physical basis for the transfer of genetic information was not known. Genes of fundamental importance do not tolerate changes (mutations) that compromise function. As a result, deleterious mutations do not accumulate in any substantial number. All living organisms have elaborate cellular systems that can recognize and eliminate faults in the integrity of DNA and genes (DNA repair). Mechanisms exist to sacrifice a cell by programmed cell death (apoptosis) if the defect cannot be successfully repaired.
Genetics in Medicine
Human genetic diseases and normal variations can be placed into one of five categories:
o single gene disorders (diseases or traits where the phenotypes are largely determined by the action, or lack of action, of mutations at individual loci);
o multifactorial traits (diseases or variations where the phenotypes are strongly influenced by the action of mutant alleles at several loci acting in concert);
o chromosomal abnormalities (diseases where the phenotypes are largely determined by physical changes in chromosomal structure – deletion, inversion, translocation, insertion, rings, etc., in chromosome number – trisomy or monosomy, or in chromosome origin – uniparental disomy);
o mitochondrial inheritance (diseases where the phenotypes are affected by mutations of mitochondrial DNA); and
o diseases of unknown etiology that seem to “run in families.”
About 1% of the approximately 4 million annual live births in the
Mendelian traits, or single gene disorders, fall into 5 categories or modes of inheritance based on where the gene for the trait is located and how many copies of the mutant allele are required to express the phenotype:
o autosomal recessive inheritance (the locus is on an autosomal chromosome and both alleles must be mutant alleles to express the phenotype)
o autosomal dominant inheritance (the locus is on an autosomal chromosome and only one mutant allele is required for expression of the phenotype)
o X-linked recessive inheritance (the locus is on the X chromosome and both alleles must be mutant alleles to express the phenotype in females)
o X-linked dominant inheritance (the locus is on the X chromosome and only one mutant allele is required for expression of the phenotype in females), and
o mitochondrial inheritance (the locus is on the mitochondrial “chromosome”).
Mendel based his laws on mathematical probabilities that allowed predictions of resulting phenotypes when certain crosses were made in the garden pea. When he published in 1866, the discovery of the chromosomal basis of inheritance (meiosis and gametogenesis) by Sutton, Boveri, and others was still a generation away. Therefore, there was no physical basis for explaining the Mendelian segregation ratios. The discoveries of Sutton, Boveri, and others allowed a reexamination of Mendel’s apparently forgotten publication. In 1900, Correns, DeVries, and Tschermak, all independently “rediscovered” Mendel’s laws of segregation, and by 1902 the first human Mendelian “inborn error of metabolism”, alcaptonuria, was found by Sir Archibald Garrod. Mendel’s laws are grounded in the chromosomal movements in meiosis, gametogenesis, and fertilization. Understanding the fundamental processes of cell division is the key to understanding Mendelian genetics.
A disease is genetically determined if it is mainly or exclusively caused by disorders in the genetic program of cells and tissues. However, most disease processes result from environmental influences interacting with the individual genetic makeup of the affected individual. These are multigenic or multifactorial diseases. They include many relatively common chronic diseases, e.g., high blood pressure, hyperlipidemia, diabetes mellitus, gout, psychiatric disorders, and certain congenital malformations.
Another common category is cancer, a large, heterogeneous group of nonhereditary genetic disorders resulting from mutations in somatic cells.
Chromosomal aberrations are also an important category. Thus, all medical specialties need to incorporate the genetic foundations of their discipline. As a rule, the genetic origin of a disease cannot be recognized by familial aggregation. Instead, the diagnosis must be based on clinical features and laboratory data. Owing to new mutations and small family size in developed countries, genetic disorders usually do not affect more than one member of a family. About 90% occur isolated within a family. Since genetic disorders affect all organ systems and age groups, and frequently go unrecognized, their contribution to the causes of human diseases appears smaller than it actually is. Genetically determined diseases are not a marginal group, but make up a substantial proportion of diseases. More than one-third of all pediatric hospital admissions are for diseases and developmental disorders that, at least in part, are caused by genetic factors. The total estimated frequency of genetically determined diseases of different categories in the general population is about 3–5% (see Table 1). The large number of individually rare genetically determined diseases and the overlap of diseases with similar clinical manifestations but different etiology cause additional diagnostic difficulties. This principle of genetic or etiological heterogeneity has to be taken into account when a diagnosis is made, to avoid false conclusions about the genetic risk.
Table 1. Categories and frequency of genetically determined diseases
|
Category of disease |
Frequency per 1000 individuals |
|
Monogenic diseases total |
5–17 |
|
Autosomal dominant |
2–10 |
|
Autosomal recessive |
2–5 |
|
X-chromosomal |
1–2 |
|
Chromosome aberrations |
5–7 |
|
Multifactorial disorders |
70–90 |
|
Somatic mutations (cancer) |
200–250 |
|
Congenital malformations |
20–25 |
|
|
Total 300–400 |
Molecular Diagnosis.
From a cell sample much can DNA diagnosis can be obtained using a number of different techniques. Many different types of mutations can be detected easily with new molecular technologies. Point mutations can be detected a variety of ways; 1) RFLP analysis or Allele specific oligo hybridization. Deletions, duplications or insertions can be detected using Southern Blot analysis or PCR analysis. Chromosome aberrations can be detected using in situ hybridization. The techniques for these methods are outlined here.
Methodologies used to test for genetic variants
I. Karyotype Analysis of Human Chromosomes
1. Karyotype preparation and analysis
Cells (from blood, amniotic fluid, etc) are grown in vitro (in a cell culture dish) to increase their number
Cell division is then arrested in metaphase with colchicine (prevents mitotic spindle from forming)
Cells are centrifuged and lysed to release chromosomes
Chromosomes are stained, photographed, and grouped by size and banding patterns
This is a photograph of the 46 human chromosomes in a somatic cell, arrested in metphase. Can you see that they are duplicated sister chromatids?
2. Normal male karyotype (a Cytogeneticist has lined these chromosomes up, matching homologues up and arranging them by size)
3. Normal female karyotype
II. Alterations in chromosome number:
Nondisjunction occurs when either homologues fail to separate during anaphase I of meiosis, or sister chromatids fail to separate during anaphase II. The result is that one gamete has 2 copies of one chromosome and the other has no copy of that chromosome. (The other chromosomes are distributed normally.)
If either of these gametes unites with another during fertilization, the result is aneuploidy (abnormal chromosome number)
A trisomic cell has one extra chromosome (2n +1) = example: trisomy 21. (Polyploidy refers to the condition of having three homologous chromosomes rather then two)
A monosomic cell has one missing chromosome (2n – 1) = usually lethal except for one known in humans: Turner’s syndrome (monosomy XO).
The frequency of nondisjunction is quite high in humans, but the results are usually so devastating to the growing zygote that miscarriage occurs very early in the pregnancy.
If the individual survives, he or she usually has a set of symptoms – a syndrome – caused by the abnormal dose of each gene product from that chromosome.
1. Human disorders due to chromosome alterations in autosomes (Chromosomes 1-22). There only 3 trisomies that result in a baby that can survive for a time after birth; the others are too devastating and the baby usually dies in utero.
A. Down syndrome (trisomy 21): The result of an extra copy of chromosome 21. People with Down syndrome are 47, 21+. Down syndrome affects 1:700 children and alters the child’s phenotype either moderately or severely:
characteristic facial features, short stature; heart defects
susceptibility to respiratory disease, shorter lifespan
prone to developing early Alzheimer’s and leukemia
often sexually underdeveloped and sterile, usually some degree of mental retardation.
Down Syndrome is correlated with age of mother but can also be the result of nondisjunction of the father’s chromosome 21.
Karyotype of a boy with Down Syndrome:
B. Patau syndrome (trisomy 13): serious eye, brain, circulatory defects as well as cleft palate. 1:5000 live births. Children rarely live more than a few months.
C. Edward’s syndrome (trisomy 18): almost every organ system affected 1:10,000 live births. Children with full Trisomy 18 generally do not live more than a few months.
2. Nondisjunction of the sex chromosomes (X or Y chromosome): Can be fatal, but many people have these karyotypes and are just fine!
A. Klinefelter syndrome: 47, XXY males. Male sex organs; unusually small testes, sterile. Breast enlargement and other feminine body characteristics. Normal intelligence.
B. 47, XYY males: Individuals are somewhat taller than average and often have below normal intelligence. At one time (~1970s), it was thought that these men were likely to be criminally aggressive, but this hypothesis has been disproven over time.
3. Trisomy X: 47, XXX females. 1:1000 live births – healthy and fertile – usually cannot be distinguished from normal female except by karyotype
4. Monosomy X (Turner’s syndrome): 1:5000 live births; the only viable monosomy in humans – women with Turner’s have only 45 chromosomes!!! XO individuals are genetically female, however, they do not mature sexually during puberty and are sterile. Short stature and normal intelligence. (98% of these fetuses die before birth)
III. Alterations in chromosome structure:
Sometimes, chromosomes break, leading to 4 types of changes in chromosome structure:
1. Deletion: a portion of one chromosome is lost during cell division. That chromosome is now missing certain genes. When this chromosome is passed on to offspring the result is usually lethal due to missing genes.
Example – Cri du chat (cry of the cat): A specific deletion of a small portion of chromosome 5; these children have severe mental retardation, a small head with unusual facial features, and a cry that sounds like a distressed cat.
2. Duplication: if the fragment joins the homologous chromosome, then that region is repeated
Example – Fragile X: the most common form of mental retardation. The X chromosome of some people is unusually fragile at one tip – seen “hanging by a thread” under a microscope. Most people have 29 “repeats” at this end of their X-chromosome, those with Fragile X have over 700 repeats due to duplications. Affects 1:1500 males, 1:2500 females.
3. Translocation: a fragment of a chromosome is moved (“trans-located”) from one chromosome to another – joins a non-homologous chromosome. The balance of genes is still normal (nothing has been gained or lost) but can alter phenotype as it places genes in a new environment. Can also cause difficulties in egg or sperm development and normal development of a zygote. Acute Myelogenous Leukemia is caused by translocation.
Restriction enzymes
Restriction enzymes are proteins that scan the DNA for specific sequences, usually palindromic sequences of 4 to 8 nucleotides, and then cleave both strands at that position. See Figure 1. There are many different restriction enzymes that can be used. To detect variation in DNA sequence restriction enzyme digestion can be used. Variation in the DNA sequence that gives rise to the creation or destruction of a restriction enzyme digestion site is called a restriction fragment length polymorphism (RFLP). Humans have two copies of each gene, except for those genes on the X and Y chromosomes in males. This must be kept in mind when doing RFLP analysis. In each sample there may be two alleles.
Southern Blot Analysis
This technique uses a Southern blot technology to identify the RFLP. Southern blots are the transfer of complex DNA to a solid state medium and then probing with a labeled probe to identify the DNA fragments of interest. The result is usually a autoradiograph with bands. This technique can usually be replaced with a PCR derivative .
PCR
This technique has revolutionized the DNA testing. It is based on the principal of DNA replication. Small pieces of DNA are replicated between oligonucleotide primers. See Figure 4-10. One can amplify any piece of the human genome with the appropriate primers and the amplified DNA can be used to test for genetic variation. Oligonucleotides are annealed to the target DNA and syntesis is carried out by a thermostabile polymerase. The PCR cycle is the denaturation of target DNA, the annealing of the oligos, and the synthesis of the new DNA. This cycle is repeated 30 times to give over 1 billion copies of the DNA molecule to use in an analysis.
Due to DNA sequence variation RFLPs can be used to map specific positions in the genome. RFLPs can be used to follow a specific gene through a family due to the linkage. In addition an RFLP can be diagnostic of a specific condition such as sickle cell anemia. See Figure 4
This mutation destroys an MstII site in the gene and can be used as a diagnostic marker for the disease allele. See Figure 5.
RFLP does not always arise from single nucleotide polymorphism due to a restriction site. Occasionally the disease allele is due to a deletion of DNA causing the DNA fragment to loose a restriction site. See Figure 6.
Allele Specific Oligos analysis
Allele specific oligos can be used to identify mutations in genes where the mutation is known. It is done with specific oligos that are complementary to each of the alleles to be detected. See Figure 4–
This technique can be converted to a PCR derivative where specific amplication of each allele is possible with a specific pair of PCR primers. The result are shown in Figure 10
Figure 10..Allele specific PCR. Oligo primer pairs are use to specifically amplify the wildtype and mutant alleles. Results are show on gel.
PCR detection of triplet repeat expansion.
Triplet repeats are found in a number of human disorders such as
References
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
9. http://intranet.tdmu.edu.ua/data/kafedra/internal/index.php?&path=pediatria2/classes_stud
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 (
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
