METHODS OF HUMAN GENETICS. CHROMOSOMAL AND MOLECULAR DISEASES.

 

DETECTING DISORDERS

Based on family history, some people know that they are at risk for transmitting certain genetic disorders. These people often want to determine whether they are carriers of these disorders before they try to have children. Parents can learn about their prospects for having healthy children based on the results of genetic tests done either before or during pregnancy.

Identifying Carriers

 Several methods are used to identify carriers of genetic disorders. Measuring enzyme levels in the blood is a way to detect carriers of a disorder such as Tay-Sachs disease. In Tay-Sachs disease, complex lipids accumulate in the brain, leading to death in early childhood. The disorder is found mostly in people of Eastern European Jewish descent. It is inherited as an autosomal recessive trait. The disorder is caused by a missing enzyme in the brain cells.

 Carriers of Tay-Sachs are heterozygous for the trait. In these people, the one dominant allele codes for the production of enough enzyme so that the disorder does not develop. However, the enzyme level in carriers is lower than that iormal individuals, who are homozygous for the dominant allele. Therefore, carriers can be identified easily by a blood test. If two carriers decide to have a child, there is a 25-percent chance that the child will inherit two recessive alleles.

 For some genetic disorders, the DNA sequences of both the normal gene and the mutated gene are known. Thus it is possible to compare the DNA of a person thought to be a carrier with the DNA iormal individuals. The comparison is done using a DNA probe. A DNA probe is a short strand of synthetic DNA with a radioactive label. The probe has a base sequence that is complementary to the base sequence of the defective gene. DNA taken from a suspected carrier is mixed with the DNA probe. If the human DNA contains the defective gene, the probe will pair with it because the base sequences are complementary. The human DNA becomes radioactive, and the person is then known to be a carrier for the disorder. Sickle-cell disease can be screened using a DNA probe.

 Sometimes it is difficult to identify the DNA that is responsible for a genetic disorder. If another known USA sequence is usually present in affected individuals, geneticists assume that the known sequence lies close to the faulty gene. The known DNA that lies close to the disease-causing gene is called a genetic marker. Within a small margin of error, the presence of the marker is an indication of the presence of the disease-causing gene. Huntington disease is usually detectable by use of a genetic marker. This newly developed test will allow people to determine if they will develop Huntington disease before they risk passing it on to their children.

 

Finding Disorders before birth

         During the past two decades, techniques have been developed for examining a fetus, or developing human, after 3 months of pregnancy. Using these techniques, doctors can determine whether a fetus has certain abnormal conditions. However, some of these techniques present a small risk to the fetus. Thus they are done only if there is reason to suspect a problem. Ultrasound and Fetoscopy In ultrasound, high-frequency sound waves are reflected off the fetus. Special equipment translates these waves into a picture on a screen. An image resulting from an ultrasound scan. Ultrasound shows the size and position of a fetus. Certain structural abnormalities, such as an improperly formed heart, also can be identified. With this information, doctors can be prepared to perform corrective surgery immediately after the baby’s birth, increasing its chance of survival.

 

Ultrasound is useful for overall structural information. However, it is sometimes necessary to have a closer look at a particular fetal structure. In fetoscopy, a technique used less commonly than ultrasound, a slender tube with a light on the end is inserted into the womb. The fetus is viewed directly. Blood and cell samples can be taken during fetoscopy and analyzed for genetic abnormalities.

 Amniocentesis and Chorionic Villus Sampling Some gene and chromosome disorders can be detected by a technique called amniocentesis. In this technique, a small amount of the fluid surrounding the fetus is carefully removed with a needle through the pregnant woman’s abdomen. The fluid contains cells shed by the developing fetus. These cells can be grown in a sterile container until there are enough of them to prepare a karyotype. The karyotype shows whether the cells of the fetus contain any abnormal, missing, or extra chromosomes. The karyotype also reveals the sex of the fetus. Amniocentesis is performed routinely on pregnant women who are over 35. These women have a higher risk of having a child with Down syndrome.

Amniocentesis cannot be performed until the fourth or fifth month of pregnancy, although some researchers are experimenting with doing the procedure as early as the twelfth week of pregnancy. Another technique, called chorionic villus sampling, or CVS, provides much of the same information as amniocentesis. In CVS, a small piece of one of the membranes that surround the fetus can be removed. This membrane sample is made of cells that are genetically identical to those of the fetus. These cells can be karyotyped and tested within a day. The procedure can be done after only 9 weeks of pregnancy.

 

Another prenatal test measures a substance called alpha-fetoprotein (AFP). This substance is normally made by the fetus, and appears in the mother’s bloodstream at known levels throughout pregnancy. At 15 weeks, AFP is measured by a simple blood test. A level much above normal may mean that the fetus has an open spine (spina bifida) or a condition called water-on-the brain. A level below normal may indicate Down syndrome. However, the test is often invalid because the AFP levels may appear abnormal if the due date for the pregnancy is miscalculated, or if twins are present. Other factors that may affect the AFP level are diabetes, race, and obesity.

Genetic Counseling

         Genetic counseling is a relatively new profession. By studying the medical histories, or pedigrees, of couples and their families, genetic counselors help parents to determine whether their risk of having a child with a genetic abnormality is higher than average. High-risk groups include people who already have a child with a genetic defect, those with a family history of genetic disease, and women over age 35. Women over age 35 are more likely to produce eggs with extra or missing chromosomes. Since those cells have been present since birth, they may have been damaged over time by chemicals, radiation, or viruses. Men over age 35 are not at higher risk for fathering children with genetic defects, because their reproductive cells are continually produced over time.

 

GENE FREQUENCIES IN NATURE

 When considering Darwin’s theory that evolution is a progressive series of adaptive changes brought about by natural selection, it is best to start by looking at the raw material available for the selective process – the genetic variation present among individuals within a species from which natural selection chooses the best-suited alleles.

A group of individuals that live together, a natural population, can contain among its members a great deal of genetic variation. This is true not only of humans but of all organisms. How much variation? Biologists have looked at many different genes in an effort to answer this question.

Blood groups. Chemical analysis has revealed the existence of more than 30 different blood group genes in humans, in addition to the ABO locus. At least a third of these genes are routinely found to be present in several alternative allelic forms in human populations. In addition to these, more than 45 additional variable genes are also known that encode proteins in human blood cells and plasma, but which are not considered to define blood groups. Thus there are more than 75 genetically variable genes in this one system alone.

 Enzymes. Alternative alleles of the genes specifying particular enzymes are easy to distinguish. The differences in their nucleotide sequences alter the ways in which the proteins specified by these alleles behave in simple physical tests. One of the most popular of these is to measure how fast the alternative proteins migrate in an electric field (a process called electrophoresis). A great deal of variation is found at enzyme-specifying loci. About 5% of the loci of a typical human are heterozygous. That is, if you picked an individual at random, and in turn selected one of the genes of that individual at random, the chances are 1 in 20 (5%) that the gene you selected would be heterozygous in that individual.

Considering the entire human genome, it is fair to say that almost all people differ from one another; this is also true of other organisms as well, except for those that reproduce without genetic recombination. Iature, genetic variation is the rule.

 How many of the loci in a given population will include more than one allele at a frequency greater than that which would be associated with mutation alone? In other words, what proportion of the loci exhibit genetic polymorphism? The extent of such variation was not even suspected more than 20 years ago, until modern techniques made it possible to discover many more polymorphic loci than could be detected on the basis of their external, physical effects (phenotype). As we now know, most populations of insects and plants are polymorphic at more than half of their loci, although vertebrates are somewhat less polymorphic. Such high levels of genetic variability provide ample supplies of raw material for evolution.

 

POPULATION GENETICS

In the early part of this century the science of genetics contributed relatively little to our understanding of the process of evolution. At first, geneticists were involved primarily with understanding the actions of individual genes. Evolutionists in turn could not understand how such observations would have a bearing on the evolution of a complex structure, such as an eye.

The gap between the geneticists and the evolutionists finally started to close in the 1920s with the development of the field of population genetics, simply defined as the study of the properties of genes in populations. At that time, scientists began to formulate a comprehensive theory of how alleles behave in populations and the ways in which changes in allele frequencies lead to evolutionary change. The most fundamental model in the field of population genetics, the Hardy-Weinberg principle, was developed in the early years of this century; all other aspects of this synthetic field can be viewed in relation to it.

 

The Hardy-Weinberg Principle

Genetic variation withiatural populations was a puzzle to Darwin and his contemporaries. The way in which meiosis produces genetic segregation among the progeny of a hybrid had not yet been discovered. The theories of the time predicted that dominant alleles should eventually drive recessive ones out of a population, thus eliminating any genetic variation. Selection, they thought, should favor an optimal form.

The solution to the puzzle of why genetic variation persists was developed independently and published almost simultaneously in 1908 by G.H. Hardy, an English mathematician, and G.Weinberg, a German physician. They pointed out that in a large population in which there is random mating, and in the absence of forces that change the proportions of the alleles at a given locus (these forces will be discussed below), the original proportions of the genotypes will remain constant from generation to generation. Dominant alleles do not in fact replace recessive ones. Because their proportions do not change, the genotypes are said to be in Hardy-Weinberg equilibrium.

In algebraic terms, the Hardy-Weinberg principle is written as an equation. Its form is what is known as a binomial expansion. For a gene with two alternative alleles, which we will call A and a, the equation looks like this:

(p + q)² = p² + 2pq + q² , where

p² – individuals homozygous for allele A,

2pq – individuals heterozygous with alleles A + a,

q² – individuals homozygous for allele a.

In statistics, frequency is defined as the proportion of individuals falling within a certain category in relation to the total number of individuals being considered. Thus, in a population of 100 cats, with 84 black and 16 white cats, the respective frequencies would be 0.84 (or 84%) and 0.16 (or 16%). In the algebraic terms of this equation, the letter p designates the frequency of one allele, the letter q the frequency of the alternative allele. By convention, the more common of the two alleles is designated p), the rarer allele q. Because there are only two alleles, p and q must always equal 1.

 If we assume that the white cats were homozygous recessive for a gene that we might designate b, and the black cats were therefore either homozygous dominant BB or heterozygous Bb, we can calculate the frequencies of the two alleles in the population from the proportion of black and white individuals. If p² = 0.16, p, the frequency of the allele B, therefore equals 0.6. In addition to the 16 white cats, which have a bb genotype, there are 2pq, or 2 X 0.6 X 0.4 X 100 (the number of individuals in the total population), or 48 heterozygous individuals. The heterozygous individuals have the Bb genotype. We can also calculate easily that there are p² = (0.6)², or 36 homozygous dominant BB individuals.

 We have assumed that the union of sperm and egg in these cats is random, so that all combinations of b and B alleles are equally likely. For this reason, the alleles are, in effect, mixed randomly and represented in the next generation in proportion to their original representation; there is no inherent reason for them to change in frequency from one generation to the next. Each individual in each generation has a 0.6 chance of receiving a B allele, and a 0.4 chance of receiving a b allele.

 In the next generation, therefore, the chance of combining two B alleles is 0.36 (that is, 0.6 X 0.6), and approximately 36% of the individuals in the population will continue to have the BB genotype. The frequency of bb individuals (0.4 X 0.4) will continue to be about 16%, and the frequency of Bb individuals will be 2 X 0.6 X 0.4, or approximately 48%. Phenotypically, there will still be approximately 84 black individuals (with either BB or Bb genotypes) and 16 white individuals (with the bb genotype) in the population.

 This simple relationship has proved extraordinarily useful in assessing actual situations. As an example, consider the recessive allele that is responsible for the serious human disease cystic fibrosis. This allele is present in white North Americans at a frequency of about 22 per 1000 individuals, or 0.022. What proportion of white North Americans, therefore, is expected to express this trait? The frequency of double recessive individuals (q²) is expected to be 0.022 X 0.022, or 1 in every 2000 individuals. What proportion is expected to be heterozygous carriers? If the frequency of the recessive allele q is 0.022, then the frequency of the dominant allele p must be 1 – 0.022, or 0.978. The frequency of heterozygous individuals (2pq) is thus expected to be 2 X 0.978 X 0.022, or 43 in every 1000 individuals.

 How valid are these calculated predictions? For many genes, they prove to be very accurate. Most human populations, for example, are large and effectively random-mating, and so are similar to the “ideal” population envisioned by Hardy and Weinberg. As we will see, however, for some genes the calculated predictions do not match the actual values. The reasons they do not do so tell us a great deal about evolution.

 The Hardy-Weinberg principle states that in a large population mating at random and in the absence of other forces that would change the proportions of the different alleles at a given locus, the process of sexual reproduction (meiosis and fertilization) alone will not change these proportions.

 

PLASMIDS AND THE NEW GENETICS

         In 1980 geneticists succeeded for the first time in introducing a human gene, the one that encodes the protein interferon, into a bacterial cell. Interferon is a rare protein that increases human resistance to viral infection and is difficult to purify in any appreciable amount. It may prove to be the basis of a useful therapy against cancer. This possibility has been difficult to explore, however, since the purification of substantial amounts of interferon required for large-scale clinical testing would, until recently, have been prohibitively expensive. An inexpensive way to produce interferon was needed, and introducing the gene responsible for its production into a bacterial cell made this possible.

 The bacterial cell that had acquired the human interferon gene proceeded to produce interferon at a high rate, and to grow and divide. Soon millions of bacterial cells were in the culture, all of them descendants of the original bacterial cell that had the human interferon gene, and all of them furiously producing interferon. This procedure of producing a line of genetically identical cells from a single “altered” cell is called cloning, had succeeded in making every cell in the culture a miniature factory for the production of human interferon. In a similar way the successful cloning of insulin was commercially significant, providing the basis for producing large amounts of a clinically important drug for relatively little expense. Cloning also has considerable theoretical significance. Such molecular techniques can and will be used increasingly to manipulate genes, and by doing so, enable us to learn more about them. This interferon experiment and others like it mark the beginning of a new genetics, the birth of genetic engineering.

 Genetic engineering is based on the ability to cut up DNA into recognizable pieces and to rearrange these pieces in different ways. In the experiment just described the gene segment carrying the interferon gene was inserted into a plasmid, which brought the inserted gene in with it when it infected the bacterial cell. Most other genetic engineering approaches have used the same general strategy of carrying the gene of interest into the target cell by first incorporating the gene into an infective plasmid or virus.

 

The success of the initial step in a genetic engineering experiment is the key to the whole procedure. As you might expect, success depends on being able to cut up the source DNA (human DNA in the interferon experiment, for example) and the plasmid DNA in such a way that the desired fragment of source DNA can be spliced permanently into the plasmid genome. This cutting is performed by a special kind of enzyme called a restriction endonuclease. These restriction enzymes are able to recognize and cleave specific sequences of nucleotides in a DNA molecule. They are the basic tools of genetic engineering. There are two classes of restriction endonucleases, called Type I and Type II. Type I nucleases make cuts at random locations, and are not useful as tools in genetic engineering. Type II nucleases recognize specific sequences. It is this second class that are commonly referred to as restriction enzymes and utilized in genetic engineering procedures.

 

RESTRICTION ENZYMES

         Scientific discoveries often have their origins in odd little crannies – seemingly unimportant areas that receive little attention by researchers before their general significance is appreciated. In the example we are considering now, the particular obscure topic was the warfare that takes place between bacteria and viruses.

 Most organisms iature eventually evolve means of defending themselves from predators, and bacteria are no exception to this rule. Among the natural enemies of bacteria are the viruses called bacteriophages, which infect bacteria, multiply within them, and eventually burst the bacterial cell, releasing thousands of viral particles. For a bacterium, a virus is a potentially lethal adversary. As a result of natural selection, those bacterial individuals that can somehow resist viral infection will be favored and leave more progeny, on the average, than those, which lack such means. Some bacteria have powerful weapons against viruses: enzymes that chop up the foreign viral DNA as soon as it enters the bacterial cell without harming the bacterial DNA at all. When viruses insert their DNA into a bacterial cell that is protected in this way, the viral DNA is immediately attacked by these enzymes, called restriction endonucleases, and degraded. Why is the DNA of the bacteria not also degraded by the restriction enzymes? Because the bacterial cell has modified its own DNA in such a way that the restriction enzymes do not recognize it as DNA.

 Restriction endonucleases recognize specific nucleotide sequences within a DNA strand, bind to DNA strands at sites where these sequences occur, and cleave the bound strand of DNA at a specific place in the recognition sequence. In this way they cut up DNA. Other bacterial enzymes called methylases recognize the same bacterial DNA sequences, bind to them, and add methyl (–CH₃) groups to the nucleotides. When the recognition sites of bacterial DNA have been modified with methyl groups in this way, they are no longer recognized by the restriction enzymes. Consequently the bacterial DNA is protected from being degraded. Viral DNA, on the other hand, is not protected because it has not been methylated.

 The sequences that restriction enzymes recognize are typically tour to six nucleotides long and symmetrical. Their symmetry is of a special kind, called twofold rotational symmetry. The nucleotides at one end of the recognition sequence are complementary to those at the other end, so that the two strands of the duplex have the same nucleotide sequence running in opposite directions for the length of the recognition sequence. This arrangement has two consequences. The first is of great importance to the bacteria; the second is of little significance in the bacterial systems, but of paramount importance to us:

 1. Because the same recognition sequence occurs on both strands of the DNA duplex (running in opposite directions), the restriction enzyme is able to recognize and cleave both strands of the duplex, effectively cutting the DNA duplex in half. This ability to chop across both strands is almost certainly the reason that restriction enzymes have evolved in such a way that they specifically recognize nucleotide sequences with twofold rotational symmetry – it lets them use one sequence to bind both strands.

 2. Because the position of the bond cleaved by a particular restriction enzyme is typically not in the center of the recognition sequence to which it binds* and because the sequence is running in opposite directions on the two strands, the sites at which the two strands of a duplex are cut are offset from one another. An example of being offset can be illustrated by taking your two hands, one palm up and the other palm down, and fitting the little fingers and ring fingers together. After cleavage the two fragments of DNA duplex each possess a short single strand a few nucleotides long dangling from the end. The two single-stranded tails are complementary to one another.

 A few restriction enzymes are known in which the cleavage position is in the center of a 4 or 6-nucleotide sequence. This results in fragments without dangling single-stranded ends – “blunt” ends. Such restriction enzymes do not generate fragments that spontaneously reassociate, and are used in genetic engineering procedures to prevent such reassociation from occurring.

 

 There are hundreds of different bacterial restriction enzymes, recognizing a wide variety of specific four-to six-nucleotide sequences. Every cleavage by a given kind of restriction enzyme takes place at the same recognition sequence. By chance, this sequence will probably occur somewhere in any given sample of DNA so that a restriction endonuclease will cut DNA from any source into fragments. Each of these fragments will have the dangling sets of complementary nucleotides (sometimes called “sticky ends”) characteristic of that endonuclease. Because the two single-stranded ends produced at a cleavage site are complementary, they can pair with each other.

Once they have done so, the two strands can then be joined back together with the aid of a sealing enzyme called a ligase, which reforms the phosphodiester bonds. This latter property makes restriction endonucleases the invaluable tools of the genetic engineer: any two fragments produced by the same restriction enzyme can be joined together. Fragments of elephant and ostrich DNA cleaved by the same bacterial restriction enzyme can be joined to one another just as readily as can two bacterial fragments because they have the same complementary sequences at their ends.

 

A restriction enzyme cleaves DNA at specific sites, generating in each case two fragments whose ends have one strand of the duplex longer than the other. Because the tailing strands of the two cleavage fragments are complementary iucleotide sequence, any pair of fragments produced by the same enzyme, from any DNA source, can be joined together.

 

CONSTRUCTING CHIMERIC GENOMES

         A chimera is a mythical creature with the head of a lion, the body of a goat, and the tail of a serpent. No such chimera ever existed iature. Human beings, however, have made them – not the lion-goat-snake variety, but chimeras of a more modest kind.

 The first actual chimera was a bacterial plasmid that American geneticists Stanley Cohen and Herbert Boyer made in 1973. Cohen and Boyer used a restriction endonuclease to cut up a large bacterial plasmid called a resistance transfer factor. From the resulting fragments, they isolated one fragment 9000 nucleotides long, which contained both the sequence necessary for replicating the plasmid – the replication origin – and a gene that conferred resistance to an antibiotic – tetracycline.

 Because both ends of this fragment were cut by the same restriction enzyme (called Escherichia coli restriction endonuclease 1, or Eco R1), they could be joined together to form a circle, a small plasmid that Cohen dubbed pSC1O1. Cohen and Boyer used the same restriction enzyme, Eco R1, to cut up DNA that they had isolated from an adult amphibian, the African clawed toad, Xenopus laevis. They then mixed the toad DNA fragments with opened-circle molecules of pSC1O1, allowed bacterial cells to take up DNA from the mixture, and selected for bacterial cells that had become resistant to tetracycline. From among these pSC1O1-containing cells they were able to isolate ones containing the toad ribosomal RNA gene. These versions of pSC1O1 had the toad gene spliced in at the Eco R1 site. Instead of joining to one another, the two ends of the pSC1O1 plasmid had joined to the two ends of the toad DNA fragment that contained the ribosomal RNA gene.

 The pSC1O1 containing the toad ribosomal RNA gene is a true chimera. It is an entirely new creature that never existed iature and would never have evolved there. It is a form of recombinant DNA, a DNA molecule created in the laboratory by molecular geneticists who joined together bits of several genomes into a novel combination.

 

The first recombinant genome produced by human genetic engineering was a bacterial plasmid into which an amphibian ribosomal RNA gene was inserted in 1973.

The insertion of fragments of foreign DNA into bacterial cells by carrying them into the cells piggyback in plasmids or viruses has become common in molecular genetics. Newer-model plasmids with exotic names, such as pBR322, can be induced to make hundreds of copies of themselves and thus of the foreign genes that are included in them within such bacterial cells. Even easier entry into bacterial cells can be achieved by inserting the foreign DNA fragment into the genome of a bacterial virus, such as lambda virus, instead of into a plasmid. The infective genome that harbors the foreign DNA and carries it into the target cell is called a vector, or vehicle. Not all vectors have bacterial targets. Animal viruses, for example, have been used as vectors to carry bacterial genes into monkey cells. Animal genes have even been carried into plant cells by using methods of this kind.

 There has been considerable discussion about the potential danger of inadvertently creating an undesirable life-form in the course of a recombinant DNA experiment. What if one fragmented the DNA of a cancer cell and then incorporated the fragments at random into viruses that were propagated within bacterial cells? Might there not be a danger that among the resulting bacteria there could be one capable of constituting an infective form of cancer?

 

 Even though most recombinant DNA experiments are not dangerous, such concerns are real and need to be taken seriously. Both scientists and individual governments monitor these experiments to detect and forestall any hazard of this sort. Experimenters have gone to considerable lengths to establish appropriate experimental safeguards. The bacteria used in many recombinant DNA experiments, for example, are unable to live outside of laboratory conditions; many of them are obligate anaerobes, poisoned by oxygen. Decidedly dangerous experiments such as cancer cell shotgun experiments (in which genomes of cancer cells are cleaved randomly and the fragments inserted into plasmids and screened) are prohibited.

 

GENETIC ENGINEERING

         The movement of genes from one organism to another, is often referred to as recombinant DNA technology or genetic engineering. While each experiment presents unique problems, all genetic engineering approaches share in common four distinct stages:

1. Cleavage. The first stage of any genetic engineering experiment is the generation of specific DNA fragments by cleavage of a genome with restriction endonuclease enzymes. Because a given six-nucleotide sequence will occur many times within a genome, restriction endonuclease cleavage will produce a large number of specific fragments, called a library. Different “libraries” of fragments may be obtained by employing enzymes that recognize different sequences. Fragments are usually compared by electrophoresis, which permits estimation of relative size.

2. Producing Recombinant DNA. The fragments of a restriction fragment library are put into plasmids or virus “vehicles,” which will later serve to carry the fragments into other cells. The key property of the recombinant vehicle is that the fragment is now replicated as part of the plasmid or virus genome. At this stage or later it is necessary to eliminate those vehicles that do NOT contain a fragment.

3. Cloning. The fragment-containing plasmid or virus vehicles are introduced into bacterial cells. Each such cell then reproduces, forming a clone of cells that all contain the fragment-bearing plasmid. Each of the cell lines is maintained separately; together they constitute a clone library of the original genome.

4. Screening. From the many clonal lines that contain fragments of the original library, it is necessary to identify those containing the specific fragment of interest, often a fragment containing a particular gene.

The fourth stage, screening, is the most critical – and difficult – experimental step.

 

 

Initial Screening of Clones

The key to a successful genetic engineering experiment lies in the strategy adopted to identify and select the desired fragment. To make this job easier, investigators usually try to eliminate from the final mix any bacterial cells that do not contain a plasmid or virus vehicle and any vehicles that do not contain a fragment from the original library. They do this by making use of genes that confer on bacteria resistance to antibiotics, chemicals such as penicillin, tetracycline, or ampicillin, which would otherwise block bacterium growth.

A). To eliminate bacteria without a vehicle, a vehicle is employed with an antibiotic resistance gene, such as one conferring tetracycline resistance. By culturing the clones of stage 4 on a medium containing tetracycline, the investigator ensures that only bacteria resistant to this antibiotic (because they contain the vehicle) will be able to grow.

B). To eliminate bacteria with a vehicle that does not contain a fragment, a vehicle is employed that has only one restriction site for the cleavage enzyme used, a site located within a second antibiotic resistance gene, such as one conferring ampicillin resistance; by testing the clones in stage 4 individually for ampicillin resistance, it becomes possible to identify directly those clones derived from cells that have successfully taken up a fragment (they have lost their resistance to this antibiotic because one of the library fragments is now sitting within the resistance gene).

 

Finding the Gene You Want

A library of restriction fragments, such as results from stage 3 above, may contain anywhere from a few dozen to many thousand individual members, each representing a different fragment of genomic DNA. A complete Drosophila (fruit fly) library, for example, contains in excess of 40,000 different clones; a complete human library of fragments 20 kilobases long would contain 150,000 clones. To identify a single fragment containing a particular gene from within that immense clone library often requires ingenuity, but many different approaches have been used successfully.

 One of the most useful procedures for identifying a specific gene has been the Southern blot, a procedure that employs purified mRNA, or a complementary DNA copy of the mRNA (called copy DNA or cDNA), as a “probe”; among thousands of fragments, only that fragment containing the proper gene will hybridize (bind by complementary base pairing) with the probe, since only that fragment has a nucleotide sequence complementary to the mRNA’s sequence. In such a screening procedure, the fragments are spread apart by electrophoresis, and a radioactive probe (synthesized in the presence of ³²P) is “blotted” onto the resulting gel pattern, incubated, and then the excess washed off; because only the gene-containing fragment will hybridize with the probe, only that fragment produces a radioactive band on film left beneath the gel.

 

TYPES OF REPRODUCTION

Reproduction is the ability to produce new individuals of ones own kind or reproduction is the biological process, which leads to the production of the new individuals. Reproduction is the important qualitative feature of a biologic form of locomotion substance.

There are two types of reproduction (Table 6):

1. Asexual Reproduction;

2. Sexual Reproduction.

Asexual reproduction is any method of producing new individuals that does not involve the fusion of gametes or nuclei of two cells and does not involve meiosis. So, at an asexual reproduction a new organism is formed with the help of somatic cells.

 

Differences Between Asexual and Sexual Reproduction

 

Sexual Reproduction is any method of producing new individuals that involves a fusion of nuclei from different sources either from different individuals or from different organs in the same individual. So, at a sexual reproduction the specialized sex cells (gametes) are formed. They differ from each other

 

Metabolic disorders or molecular pathology are caused by mutation in the genes.

  Phenylketonuria – is recessive autosomal inherited trait caused by the lack of an enzyme phenylalanine hydroxilase needed to change one amino acid, phenylalanine, to another, tyrosine. It characterized by mental retardation, hypopigmentation of hair and skin, and mousy odour.

                             

 

Albinism (from Latin albus, “white”; see extended etymology, also called achromia, achromasia, or achromatosis) is a congenital disordercharacterized by the complete or partial absence of pigment in the skin, hair and eyes due to absence or defect of tyrosinase, a copper-containing enzyme involved in the production of melanin. Albinism results from inheritance of recessive gene alleles and is known to affect all vertebrates, including humans. While an organism with complete absence of melanin is called an albino an organism with only a diminished amount of melanin is described as albinoid.

Albinism is associated with a number of vision defects, such as photophobia, nystagmus and astigmatism. Lack of skin pigmentation makes for more susceptibility to sunburn and skin cancers. In rare cases such as Chédiak–Higashi syndrome, albinism may be associated with deficiencies in the transportation of melanin granules. This also affects essential granules present in immune cells leading to increased susceptibility to infection.

In humans, there are two principal types of albinism, oculocutaneous, affecting the eyes, skin and hair, and ocular affecting the eyes only.

Most oculocutaenous albinistic humans appear white or very pale as the melanin pigments responsible for brown, black, and some yellow colorations are not present. Ocular albinism results in pale blue eyes, and may require genetic testing to diagnose.

Because individuals with albinism have skin that entirely lacks the dark pigment melanin, which helps protect the skin from the sun’s ultraviolet radiation, their skin can burn more easily from overexposure.

 The human eye normally produces enough pigment to color the iris blue, green or brown and lend opacity to the eye. However, there are cases in which the eyes of an albinistic person appear red, pink or purple, depending on the amount of pigment present, due to the red ofretina being visible through the iris. Lack of pigment in the eyes also results in problems with vision, both related and unrelated tophotosensitivity.

Those inflicted with albinism are generally as healthy as the rest of the population (but see related disorders below), with growth and development occurring as normal, and albinism by itself does not cause mortality,^[6] although the lack of pigment blocking ultravioletradiation increases the risk of Melanomas (skins cancers) and other problems.

 

Most forms of albinism are the result of the biological inheritance of genetically recessive alleles (genes) passed from both parents of an individual, though some rare forms are inherited from only one parent. There are other genetic mutations which are proven to be associated with albinism. All alterations, however, lead to changes in melanin production in the body.

The chance of offspring with albinism resulting from the pairing of an organism with albinism and one without albinism is low. However, because organisms (including humans) can be carriers of genes for albinism without exhibiting any traits, albinistic offspring can be produced by two non-albinistic parents. Albinism usually occurs with equal frequency in both sexes.^[6] An exception to this is ocular albinism, which it is passed on to offspring through X-linked inheritance. Thus, ocular albinism occurs more frequently in males as they have a single X and Y chromosome, unlike females, whose genetics are characterized by two X chromosomes.^[12]

There are two different forms of albinism: a partial lack of the melanin is known as hypomelanism, or hypomelanosis and the total absence of melanin is known as amelanism or amelanosis.

         In physical terms, humans with albinism commonly have visual problems and need sun protection. They often face social and cultural challenges (even threats), as the condition is often a source of ridicule, discrimination, or even fear and violence. Many cultures around the world have developed beliefs regarding people with albinism.

In African countries such as Tanzania and Burundi, there has been an unprecedented rise in witchcraft-related killings of albino people in recent years, because their body parts are used in potions sold by witchdoctors. Numerous authenticated incidents have occurred in Africa during the 21st Century. For example, in Tanzania, in September 2009, three men were convicted of killing a 14-year-old albino boy and severing his legs in order to sell them for witchcraft purposes. Again in Tanzania and Burundi in 2010, the murder and dismemberment of a kidnapped albino child was reported from the courts,^[16] as part of a continuing problem. National Geographic estimates that in Tanzania a complete set of albino body parts is worth $75,000.

Another harmful and false belief is that sex with an albinistic woman will cure a man of HIV. This has led, for example in Zimbabwe, to rapes (and subsequent HIV infection).

Certain ethnic groups and insular areas exhibit heightened susceptibility to albinism, presumably due to genetic factors. These include notably the Native American Kuna and Zuni nations (respectively of Panama and New Mexico); Japan, in which one particular form of albinism is unusually common; and Ukerewe Island, the population of which shows a very high incidence of albinism.

Famous people with albinism include historical figures such as Oxford don William Archibald Spooner; actor-comedian Victor Varnado; musicians such as Johnny and Edgar Winter, Salif Keita,Winston “Yellowman” Foster, Brother Ali, Sivuca, Willie “Piano Red” Perryman; and fashion models Connie Chiu and Shaun Ross. Emperor Seinei of Japan is thought to have been an albino because he was said to have been born with white hair.

 

 

Sickle-cell anaemia – is incompletely dominant trait caused by defective type of haemoglobin called sickle-cell haemoglobin or Hb^S. Persons with genotype Hb^SHb^S have sickle-cell disease, which is characterized by sickle-shaped red blood cells, acute attacks of abdomen pain, arthralgia. Persons with genotype Hb^AHb^A are normal. Persons with genotype Hb^SHb^A have sickle-cell trait, a condition in which erythrocytes are sometimes sickle shaped.

 

Sickle-cell disease (SCD), or sickle-cell anaemia (SCA) or drepanocytosis, is a hereditary blood disorder, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells’ flexibility and results in a risk of various complications. The sickling occurs because of a mutation in the haemoglobin gene. Individuals with one copy of the defunct gene display both normal and abnormal haemoglobin. This is an example of codominance.

Life expectancy is shortened. In 1994, in the US, the average life expectancy of persons with this condition was estimated to be 42 years in males and 48 years in females, but today, thanks to better management of the disease, patients can live into their 70s or beyond.

Sickle-cell disease occurs more commonly among people whose ancestors lived in tropical and sub-tropical sub-saharan regions where malaria is or was common. Where malaria is common, carrying a single sickle-cell gene (sickle cell trait) confers a fitness. Specifically, humans with one of the two alleles of sickle-cell disease show less severe symptoms when infected with malaria.

Sickle-cell anaemia is a form of sickle-cell disease in which there is homozygosity for the mutation that causes HbS. Sickle-cell anaemia is also referred to as “HbSS”, “SS disease”, “haemoglobin S” or permutations of those names. In heterozygous people, that is, those who have only one sickle gene and one normal adult haemoglobin gene, the condition is referred to as “HbAS” or “sickle cell trait”. Other, rarer forms of sickle-cell disease are compound heterozygous states in which the person has only one copy of the mutation that causes HbS and one copy of another abnormal haemoglobin allele. They include sickle-haemoglobin C disease (HbSC), sickle beta-plus-thalassaemia (HbS/β⁺) and sickle beta-zero-thalassaemia (HbS/β⁰).

The term disease is applied because the inherited abnormality causes a pathological condition that can lead to death and severe complications. Not all inherited variants of haemoglobin are detrimental, a concept known as genetic polymorphism.

 Sickle cells in human blood: both normal red blood cells and sickle-shaped cells are present

 Sickle-cell disease may lead to various acute and chronic complications, several of which have a high mortality rate.^[

 

Sickle cell crisis

The terms “sickle cell crisis” or “sickling crisis” may be used to describe several independent acute conditions occurring in patients with sickle cell disease. Sickle cell disease results in anemia and crises that could be of many types including the vaso-occlusive crisis, aplastic crisis, sequestration crisis, haemolytic crisis and others. Most episodes of sickle cell crises last between five and seven days. “Although infection, dehydration, and acidosis (all of which favor sickling) can act as triggers, in most instances no predisposing cause is identified.”

 

Vaso-occlusive crisis

The vaso-occlusive crisis is caused by sickle-shaped red blood cells that obstruct capillaries and restrict blood flow to an organ, resulting in ischaemia,pain, necrosis and often organ damage. The frequency, severity, and duration of these crises vary considerably. Painful crises are treated with hydration, analgesics, and blood transfusion; pain management requires opioid administration at regular intervals until the crisis has settled. For milder crises, a subgroup of patients manage on NSAIDs (such as diclofenac or naproxen). For more severe crises, most patients require inpatient management for intravenous opioids; patient-controlled analgesia (PCA) devices are commonly used in this setting. Vaso-occlusive crisis involving organs such as the penis or lungs are considered an emergency and treated with red-blood cell transfusions. Diphenhydramine is sometimes effective for the itching associated with the opioid use. Incentive spirometry, a technique to encourage deep breathing to minimise the development of atelectasis, is recommended.

 

Splenic sequestration crisis

 Because of its narrow vessels and function in clearing defective red blood cells, the spleen is frequently affected. It is usually infarcted before the end of childhood in individuals suffering from sickle-cell anemia. This autosplenectomy increases the risk of infection from encapsulated organisms; preventive antibiotics and vaccinations are recommended for those with such asplenia.

Splenic sequestration crises: are acute, painful enlargements of the spleen, caused by intrasplenic trapping of red cells and resulting in a precipitous fall in hemoglobin levels with the potential for hypovolemic shock. Sequestration crises are considered an emergency. If not treated, patients may die within 1–2 hours due to circulatory failure. Management is supportive, sometimes with blood transfusion. These crises are transient, they continue for 3–4 hours and may last for one day.

Aplastic crisis

         Aplastic crises are acute worsenings of the patient’s baseline anaemia, producing pallor, tachycardia, and fatigue. This crisis is normally triggered by parvovirus B19, which directly affectserythropoiesis (production of red blood cells) by invading the red cell precursors and multiplying in them and destroying them. Parvovirus infectioearly completely prevents red blood cell production for two to three days. Iormal individuals, this is of little consequence, but the shortened red cell life of sickle-cell patients results in an abrupt, life-threatening situation. Reticulocytecounts drop dramatically during the disease (causing reticulocytopenia), and the rapid turnover of red cells leads to the drop in haemoglobin. This crisis takes 4 days to one week to disappear. Most patients can be managed supportively; some need blood transfusion.

Haemolytic crisis

 Haemolytic crises are acute accelerated drops in haemoglobin level. The red blood cells break down at a faster rate. This is particularly common in patients with co-existent G6PD deficiency.^[14]Management is supportive, sometimes with blood transfusions.

Normally, humans have Haemoglobin A, which consists of two alpha and two beta chains, Haemoglobin A2, which consists of two alpha and two delta chains and Haemoglobin F, consisting of two alpha and two gamma chains in their bodies. Of these, Haemoglobin A makes up around 96-97% of the normal haemoglobin in humans.

Sickle-cell gene mutation probably arose spontaneously in different geographic areas, as suggested by restriction endonuclease analysis. These variants are known as Cameroon, Senegal, Benin, Bantu and Saudi-Asian. Their clinical importance springs from the fact that some of them are associated with higher HbF levels, e.g., Senegal and Saudi-Asian variants, and tend to have milder disease.

In people heterozygous for HgbS (carriers of sickling haemoglobin), the polymerisation problems are minor, because the normal allele is able to produce over 50% of the haemoglobin. In peoplehomozygous for HgbS, the presence of long-chain polymers of HbS distort the shape of the red blood cell from a smooth doughnut-like shape to ragged and full of spikes, making it fragile and susceptible to breaking within capillaries. Carriers have symptoms only if they are deprived of oxygen (for example, while climbing a mountain) or while severely dehydrated. The sickle-cell disease occurs when the sixth amino acid, glutamic acid, is replaced by valine to change its structure and function; as such, sickle cell anemia is also known as E6V. Valine is hydrophobic, causing the haemoglobin to collapse in on itself occasionally. The structure is not changed otherwise. When enough haemoglobin collapses in on itself the red blood cells become sickle-shaped.

 

Distribution of the sickle-cell trait shown in pink and purple

 Historical distribution ofmalaria (no longer endemic in Europe) shown in green

 Modern distribution of malaria

The gene defect is a known mutation of a single nucleotide (see single-nucleotide polymorphism – SNP) (A to T) of the β-globin gene, which results in glutamic acidbeing substituted by valine at position 6. Haemoglobin S with this mutation is referred to as HbS, as opposed to the normal adult HbA. The genetic disorder is due to the mutation of a single nucleotide, from a GAG to GTG codon on the coding strand, which is transcribed from the template strand into a GUG codon. This is normally a benign mutation, causing no apparent effects on the secondary, tertiary, or quaternary structure of haemoglobin in conditions of normal oxygenconcentration. What it does allow for, under conditions of low oxygen concentration, is the polymerization of the HbS itself. The deoxy form of haemoglobin exposes a hydrophobic patch on the protein between the E and F helices. The hydrophobic residues of the valine at position 6 of the beta chain in haemoglobin are able to associate with the hydrophobic patch, causing haemoglobin S molecules to aggregate and form fibrous precipitates.

 The allele responsible for sickle-cell anaemia can be found on the short arm of chromosome 11. A person that receives the defective gene from both father and mother develops the disease; a person that receives one defective and one healthy allele remains healthy, but can pass on the disease and is known as a carrier. If two parents who are carriers have a child, there is a 1-in-4 chance of their child developing the disease and a 1-in-2 chance of their child being just a carrier. Heterozygotes are still able to contract malaria, but their symptoms are generally less severe.

         Due to the adaptive advantage of the heterozygote, the disease is still prevalent, especially among people with recent ancestry in malaria-stricken areas, such asAfrica, the Mediterranean, India and the Middle East. Malaria was historically endemic to southern Europe, but it was declared eradicated in the mid-20th century, with the exception of rare sporadic cases.

The malaria parasite has a complex life cycle and spends part of it in red blood cells. In a carrier, the presence of the malaria parasite causes the red blood cells with defective haemoglobin to rupture prematurely, making the plasmodium unable to reproduce. Further, the polymerization of Hb affects the ability of the parasite to digest Hb in the first place. Therefore, in areas where malaria is a problem, people’s chances of survival actually increase if they carry sickle-cell trait (selection for the heterozygote).

In the USA, where there is no endemic malaria, the prevalence of sickle-cell anaemia among blacks is lower (about 0.25%) than in West Africa (about 4.0%) and is falling. Without endemic malaria, the sickle cell mutation is purely disadvantageous and will tend to be selected out of the affected population via natural selection. However, the African American community of the USA is known to be the result of significant admixture between several African and non-African ethnic groups, and also represents the descendants of survivors of the slavery and the slave trade. Thus, a lower degree of endogamy and, particularly, abnormally high health-selective pressure through slavery may be the most plausible explanations for the lower prevalence of sickle-cell anaemia (and, possibly, other genetic diseases) among African-Americans compared to Sub-Saharan Africans. Another factor limiting the spread of sickle-cell genes in North America is the absence of cultural proclivities to polygamy, which allows affected males to continue to seek unaffected children with multiple partners.

 

 Inheritance 

Sickle-cell conditions are inherited from parents in much the same way as blood type, hair color and texture, eye colour, and other physical traits. The types of haemoglobin a person makes in the red blood cells depend on what haemoglobin genes are inherited from his parents. If one parent has sickle-cell anaemia (SS) and the other has sickle-cell trait then there is a 50% chance of a child’s having sickle-cell disease and a 50% chance of a child’s having sickle-cell trait. When both parents have sickle-cell trait a child has a 25% chance of sickle-cell disease, as shown in the diagram.

Anemia. Sickle cells are fragile. They break apart easily and die, leaving you chronically short on red blood cells. Red blood cells usually live for about 120 days before they die and need to be replaced. However, sickle cells die after only 10 to 20 days. The result is a chronic shortage of red blood cells, known as anemia. Without enough red blood cells in circulation, your body can’t get the oxygen it needs to feel energized. That’s why anemia causes fatigue.

o    Episodes of pain. Periodic episodes of pain, called crises, are a major symptom of sickle cell anemia. Pain develops when sickle-shaped red blood cells block blood flow through tiny blood vessels to your chest, abdomen and joints. Pain can also occur in your bones. The pain may vary in intensity and can last for a few hours to a few weeks. Some people experience only a few episodes of pain. Others experience a dozen or more crises a year. If a crisis is severe enough, you may need to be hospitalized.

o    Hand-foot syndrome. Swollen hands and feet may be the first signs of sickle cell anemia in babies. The swelling is caused by sickle-shaped red blood cells blocking blood flow out of their hands and feet.

o    Frequent infections. Sickle cells can damage your spleen, an organ that fights infection. This may make you more vulnerable to infections. Doctors commonly give infants and children with sickle cell anemia antibiotics to prevent potentially life-threatening infections, such as pneumonia.

Delayed growth. Red blood cells provide your body with the oxygen and nutrients you need for growth. A shortage of healthy red blood cells can slow growth in infants and children and delay puberty in teenagers.

Vision problems. Some people with sickle cell anemia experience vision problems. Tiny blood vessels that supply your eyes may become plugged with sickle cells. This can damage the retina — the portion of the eye that processes visual images.

In HbSS, the full blood count reveals haemoglobin levels in the range of 6–8 g/dL with a high reticulocyte count (as the bone marrow compensates for the destruction of sickle cells by producing more red blood cells). In other forms of sickle-cell disease, Hb levels tend to be higher. A blood film may show features of hyposplenism (target cells and Howell-Jolly bodies).

Sickling of the red blood cells, on a blood film, can be induced by the addition of sodium metabisulfite. The presence of sickle haemoglobin can also be demonstrated with the “sickle solubility test”. A mixture of haemoglobin S (Hb S) in a reducing solution (such as sodium dithionite) gives a turbid appearance, whereas normal Hb gives a clear solution.

Abnormal haemoglobin forms can be detected on haemoglobin electrophoresis, a form of gel electrophoresis on which the various types of haemoglobin move at varying speeds. Sickle-cell haemoglobin (HgbS) and haemoglobin C with sickling (HgbSC)—the two most common forms—can be identified from there. The diagnosis can be confirmed with high-performance liquid chromatography (HPLC). Genetic testing is rarely performed, as other investigations are highly specific for HbS and HbC.

An acute sickle-cell crisis is often precipitated by infection. Therefore, a urinalysis to detect an occult urinary tract infection, and chest X-ray to look for occult pneumonia should be routinely performed.

People who are known carriers of the disease often undergo genetic counseling before they have a child. A test to see if an unborn child has the disease takes either a blood sample from the fetusor a sample of amniotic fluid. Since taking a blood sample from a fetus has greater risks, the latter test is usually used.

After the mutation responsible for this disease was discovered in 1979, the U.S. Air Force required black applicants to test for the mutation. It dismissed 143 applicants because they were carriers, even though none of them had the condition. It eventually withdrew the requirement, but only after a trainee filed a lawsuit.

 

  Tay-Sach’s disease – is recessive autosomal inherited trait caused by the lack of an enzyme hexosaminidase A (Hex A) and the subsequent storage of its substrate, a glycosphingolipid, in lisosomes of the cells of the nervous system. The child having this disease is borormal, but a few moths later, due to an error mental retardation and paralysis. Ill children die between 2 and 5 years of age.

Tay–Sachs disease (also known as GM2 gangliosidosis or hexosaminidase A deficiency) is a rare autosomal recessive genetic disorder. In its most common variant (known as infantile Tay–Sachs disease), it causes a progressive deterioration of nerve cells and of mental and physical abilities that commences around six months of age and usually results in death by the age of four. The disease occurs when harmful quantities of cell membrane components known as gangliosides accumulate in the brain‘s nerve cells, eventually leading to the premature death of the cells. A ganglioside is a form of sphingolipid, which makes Tay–Sachs disease a member of the sphingolipidoses. There is no known cure or treatment.

The disease is named after the British ophthalmologist Waren Tay, who in 1881 first described a symptomatic red spot on the retina of the eye, and after the American neurologist Bernard Sachs of Mount Sinai Hospital, New York, who described in 1887 the cellular changes of Tay–Sachs disease and noted an increased disease prevalence in the Eastern European Ashkenazi Jewish population.

Research in the late 20th century demonstrated that Tay–Sachs disease is caused by a genetic mutation in the HEXA gene on (human) chromosome 15. A large number of HEXA mutations have been discovered, and new ones are still being reported. These mutations reach significant frequencies in specific populations. French Canadians of southeastern Quebec have a carrier frequency similar to that seen in Ashkenazi Jews, but carry a different mutation. Cajuns of southern Louisiana carry the same mutation that is seen most commonly in Ashkenazi Jews. HEXA mutations are rare and are most seen in genetically isolated populations. Tay–Sachs can occur from the inheritance of either two similar, or two unrelated, causative mutations in the HEXA gene.

Tay–Sachs disease is classified into several forms, which are differentiated based on the onset age of neurological symptoms.

 

Infantile Tay–Sachs disease. Infants with Tay–Sachs disease appear to develop normally for the first six months after birth. Then, as neurons become distended with gangliosides, a relentless deterioration of mental and physical abilities begins. The child becomes blind, deaf, unable to swallow, atrophied, and paralytic. Death usually occurs before the age of four.

Juvenile Tay–Sachs disease. Juvenile Tay–Sachs disease is rarer than other forms of Tay–Sachs, and usually is initially seen in children between two and ten years old. People with Tay–Sachs disease develop cognitive and motor skill deterioration, dysarthria, dysphagia, ataxia, and spasticity.^[3] Death usually occurs between the age of five to fifteen year

Adult/Late-Onset Tay–Sachs disease. A rare form of this disease, known as Adult-Onset or Late-Onset Tay–Sachs disease, usually has its first symptoms during the 30s or 40s. In contrast to the other forms, late-onset Tay–Sachs disease is usually not fatal as the effects can stop progressing. It is frequently misdiagnosed. It is characterized by unsteadiness of gait and progressive neurological deterioration. Symptoms of late-onset Tay–Sachs – which typically begin to be seen in adolescence or early adulthood – include speech and swallowing difficulties, unsteadiness of gait, spasticity, cognitive decline, and psychiatric illness, particularly a schizophrenia-like psychosis. People with late-onset Tay–Sachs may become full-time wheelchairusers in adulthood.

Until the 1970s and 1980s, when the disease’s molecular genetics became known, the juvenile and adult forms of the disease were not always recognized as variants of Tay–Sachs disease. Post-infantile Tay–Sachs was often misdiagnosed as another neurological disorder, such as Friedreich’s ataxia.

Genetics

Tay–Sachs disease is inherited in theautosomal recessive pattern, depicted above.

The HEXA gene is located on the long (q) arm of human chromosome 15, between positions 23 and 24

 

Tay–Sachs disease is an autosomal recessive genetic disorder, meaning that when both parents are carriers there is a 25% risk of giving birth to an affected child with each pregnancy. The affected child would have received a mutated copy of the gene from each parent.

Tay–Sachs results from mutations in the HEXA gene on human chromosome 15, which encodes the alpha-subunit of beta-N-acetylhexosaminidase A, a lysosomal enzyme. By 2000, more than 100 different mutations had been identified in the human HEXA gene. These mutations have included single base insertions and deletions, splice phase mutations, missense mutations, and other more complex patterns. Each of these mutations alters thegene’s protein product (i.e., the enzyme), sometimes severely inhibiting its function. In recent years, population studies and pedigree analysis have shown how such mutations arise and spread within small founder populations. Initial research focused on several such founder populations:

 

Ashkenazi Jews. A four base pair insertion in exon 11 (1278insTATC) results in an altered reading frame for the HEXA gene. This mutation is the most prevalent mutation in the Ashkenazi Jewish population, and leads to the infantile form of Tay–Sachs disease.

Cajun. The same 1278insTATC mutation found among Ashkenazi Jews occurs in the Cajun population of southern Louisiana. Researchers have traced the ancestry of carriers from Louisiana families back to a single founder couple – not known to be Jewish – that lived in France in the 18th century.

French Canadians. Two mutations, unrelated to the Ashkenazi/Cajun mutation, are absent in France but common among French Canadians living in eastern Quebec. Pedigree analysis suggests the mutations were uncommon before the late 17th century.

In the 1960s and early 1970s, when the biochemical basis of Tay–Sachs disease was first becoming known, no mutations had been sequenced directly for genetic diseases. Researchers of that era did not yet know how common polymorphisms would prove to be. The “Jewish Fur Trader Hypothesis,” with its implication that a single mutation must have spread from one population into another, reflected the knowledge at the time. Subsequent research, however, has proven that a large variety of different HEXA mutations can cause the disease. Because Tay–Sachs was one of the first genetic disorders for which widespread genetic screening was possible, it is one of the first genetic disorders in which the prevalence of compound heterozygosity has been demonstrated.

Compound heterozygosity ultimately explains the disease’s variability, including the late-onset forms. The disease can potentially result from the inheritance of two unrelated mutations in the HEXA gene, one from each parent. Classic infantile Tay–Sachs disease results when a child has inherited mutations from both parents that completely stop the biodegradation of gangliosides. Late onset forms occur due to the diverse mutation base – people with Tay–Sachs disease may technically be heterozygotes, with two differing HEXA mutations that both inactivate, alter, or inhibit enzyme activity. When a patient has at least one HEXA copy that still enables some level of hexosaminidase A activity, a later onset disease form occurs. When disease occurs because of two unrelated mutations, the patient is said to be a compound heterozygote.

         Heterozygous carriers (individuals who inherit one mutant allele) show abnormal enzyme activity, but manifest no disease symptoms. This phenomenon is called dominance; the biochemical reason for wild-type alleles’ dominance over nonfunctional mutant alleles in inborn errors of metabolism comes from how enzymes function. Enzymes are protein catalysts for chemical reactions; as catalysts, they speed up reactions without being used up in the process, so only small enzyme quantities are required to carry out a reaction. Someone homozygous for a nonfunctional mutation in the enzyme-encoding gene has little or no enzyme activity, so will manifest the abnormal phenotype. A heterozygote (heterozygous individual) has at least half of the normal enzyme activity level, due to expression by the wild-type allele. This level is normally enough to enable normal function and thus prevent phenotypic expression.

In patients with a clinical suspicion for Tay–Sachs disease, with any age of onset, the initial testing involves an enzyme assay to measure the activity of hexosaminidase in serum, fibroblasts orleukocytes. Total hexosaminidase enzyme activity is decreased in individuals with Tay-Sachs as is the percentage of hexosaminidase A. After confirmation of decreased enzyme activity in an individual, confirmation by molecular analysis can be pursued. All patients with infantile onset Tay–Sachs disease have a “cherry red” macula in the retina, easily observable by a physician using an ophthalmoscope. This red spot is a retinal area that appears red because of gangliosides in the surrounding retinal ganglion cells. The choroidal circulation is showing through “red” in this foveal region where all retinal ganglion cells are pushed aside to increase visual acuity. Thus, this cherry-red spot is the only normal part of the retina; it shows up in contrast to the rest of the retina. Microscopic analysis of the retinal neurons shows they are distended from excess ganglioside storage. Unlike other lysosomal storage diseases (e.g., Gaucher disease, Niemann–Pick disease, and Sandhoff disease), hepatosplenomegaly (liver and spleen enlargement) is not seen in Tay–Sachs.

Prevention Three main approaches have been used to prevent or reduce the incidence of Tay–Sachs:

Prenatal diagnosis. If both parents are identified as carriers, prenatal genetic testing can determine whether the fetus has inherited a defective gene copy from both parents. Couples are informed and may choose to have an abortion.^[21] Chorionic villus sampling (CVS), the most common form of prenatal diagnosis, can be performed between 10 and 14 weeks of gestation.Amniocentesis is usually performed at 15-18 weeks. These procedures have risks of miscarriage of 1% or less.

 

Preimplantation genetic diagnosis. By retrieving the mother’s eggs for in vitro fertilization, it is possible to test the embryo for the disorder prior to implantation. Healthy embryos are then selected and transferred into the mother’s womb, while unhealthy embryos are discarded. In addition to Tay–Sachs disease, preimplantation genetic diagnosis has been used to prevent cystic fibrosis and sickle cell anemia among other genetic disorders.^[24]

Mate selection. In Orthodox Jewish circles, the organization Dor Yeshorim carries out an anonymous screening program so that couples with Tay–Sachs or another genetic disorder can avoid conception.

 

 

There is currently no cure or treatment for Tay–Sachs disease. Even with the best care, children with infantile Tay–Sachs disease die by the age of 4. Although experimental work is underway, no current medical treatment of the root cause yet exists. Patients receive supportive care to ease the symptoms or extend life. Infants are given feeding tubes when they cao longer swallow. Improvements in life-extending care have somewhat lengthened the survival of children with Tay–Sachs disease, but no current therapy is able to reverse or delay the disease’s progress. In late-onset Tay-Sachs, medication (e.g., lithium for depression) can sometimes control psychiatric symptoms and seizures, although some medications (e.g., tricyclic antidepressants, phenothiazines, haloperidol, and risperidone) are associated with significant adverse effects. In 2011, researchers have discovered that Pyrimethamine can increase ß-hexosaminidase activity, thus slowing down the progression of Late-Onset Tay–Sachs disease.

 

Enzyme replacement therapy

Enzyme replacement therapy techniques have been investigated for lysosomal storage disorders, and could potentially be used to treat Tay–Sachs as well. The goal would be to replace the nonfunctional enzyme, a process similar to insulin injections for diabetes. However, in previous studies the HEXA enzyme itself has been thought to be too large to pass through the specialized cell layer in the blood vessels that forms the blood–brain barrier in humans.

Researchers have also tried directly instilling the deficient enzyme hexosaminidase A into the cerebrospinal fluid (CSF), which bathes the brain. However, intracerebral neurons seem unable to take up this physically large molecule efficiently even when it is directly by them. Therefore, this approach to treatment of Tay–Sachs disease has also been ineffective so far.^[47]

Waren Tay and Bernard Sachs, two physicians, described the disease’s progression and provided differential diagnostic criteria to distinguish it from other neurological disorders with similar symptoms.

Both Tay and Sachs reported their first cases among Jewish families. Tay reported his observations in 1881 in the first volume of the proceedings of the British Ophthalmological Society, of which he was a founding member. By 1884, he had seen three cases in a single family. Years later, Bernard Sachs, an American neurologist, reported similar findings when he reported a case of “arrested cerebral development” to other New York Neurological Society members.

 Sachs, who recognized that the disease had a familial basis, proposed that the disease should be called amaurotic familial idiocy. However, its genetic basis was still poorly understood. AlthoughGregor Mendel had published his article on the genetics of peas in 1865, Mendel’s paper was largely forgotten for more than a generation – not rediscovered by other scientists until 1899. Thus, the Mendelian model for explaining Tay–Sachs was unavailable to scientists and doctors of the time. The first edition of the Jewish Encyclopedia, published in 12 volumes between 1901 and 1906, described what was then known about the disease:

It is a curious fact that amaurotic family idiocy, a rare and fatal disease of children, occurs mostly among Jews. The largest number of cases has been observed in the United States—over thirty iumber. It was at first thought that this was an exclusively Jewish disease, because most of the cases at first reported were between Russian and Polish Jews; but recently there have been reported cases occurring ion-Jewish children. The chief characteristics of the disease are progressive mental and physical enfeeblement; weakness and paralysis of all the extremities; and marasmus, associated with symmetrical changes in the macula lutea. On investigation of the reported cases, they found that neither consanguinity nor syphilitic, alcoholic, or nervous antecedents in the family history are factors in the etiology of the disease. No preventive measures have as yet been discovered, and no treatment has been of benefit, all the cases having terminated fatally.

Jewish immigration to the United States peaked in the period 1880–1924, with the immigrants arriving from Russia and countries in Eastern Europe; this was also a period of nativism (hostility to immigrants) in the United States. Opponents of immigration often questioned whether immigrants from southern and eastern Europe could be assimilated into American society. Reports of Tay–Sachs disease contributed to a perception among nativists that Jews were an inferior race. Reuter writes “that Jewish immigrants continued to display their nervous tendencies in America where they were free from persecution was seen as proof of their biological inferiority and raised concerns about the degree to which they were being permitted free entry into the US.”^[41]

In 1969, Shintaro Okada and John S. O’Brien showed that Tay–Sachs disease was caused by an enzyme defect; he also proved that Tay–Sachs patients could be diagnosed by an assay of hexosaminidase A activity.^[42] The further development of enzyme assays demonstrated that levels of hexosaminidases A and B could be measured in patients and carriers, allowing the reliable detection of heterozygotes. During the early 1970s, researchers developed protocols for newborn testing, carrier screening, and pre-natal diagnosis. By the end of 1979, researchers had identified three variant forms of GM2 gangliosidosis, including Sandhoff disease and the AB variant of GM2-gangliosidosis, accounting for false negatives in carrier testing.

Phenylketonuria (PKU) is an autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzymephenylalanine hydroxylase (PAH), rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid tyrosine. When PAH activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate (also known as phenylketone), which can be detected in the urine.

Untreated PKU can lead to mental retardation, seizures, and other serious medical problems. The mainstream treatment for classic PKU patients is a strict PHE-restricted diet supplemented by a medical formula containing amino acids and other nutrients. In the United States, the current recommendation is that the PKU diet should be maintained for life. Patients who are diagnosed early and maintain a strict diet can have a normal life span with normal mental development. However, recent research suggests that neurocognitive, psychosocial, quality of life, growth, nutrition, bone pathology are slightly suboptimal if diet is not supplemented with amino acids.

Phenylketonuria was discovered by the Norwegian physician Ivar Asbjørn Følling in 1934 when he noticed hyperphenylalaninemia (HPA) was associated with mental retardation. In Norway, this disorder is known as Følling’s disease, named after its discoverer. Dr. Følling was one of the first physicians to apply detailed chemical analysis to the study of disease. His careful analysis of the urine of two affected siblings led him to request many physicians near Oslo to test the urine of other affected patients. This led to the discovery of the same substance he had found in eight other patients. He conducted tests and found reactions that gave rise to benzaldehyde and benzoic acid, which led him to conclude that the compound contained a benzene ring. Further testing showed the melting point to be the same as phenylpyruvic acid, which indicated that the substance was in the urine. His careful science inspired many to pursue similar meticulous and painstaking research with other disorders. It was recently suggested that PKU may resemble amyloid diseases, such as Alzheimer’s disease and Parkinson’s disease, due to the formation of toxic amyloid-like assemblies of phenylalanine.

 

Blood is taken from a two-week old infant to test for phenylketonuria

 

PKU is commonly included in the newborn screening panel of most countries, with varied detection techniques. Most babies in developed countries are screened for PKU soon after birth. Screening for PKU is done with bacterial inhibition assay (Guthrie test), immunoassays using fluorometric or photometric detection, or amino acid measurement using tandem mass spectrometry (MS/MS). Measurements done using MS/MS determine the concentration of Phe and the ratio of Phe to tyrosine, both of which will be elevated in PKU.

If a child is not screened during the routine newborn screening test (typically performed 2 – 7 days after birth, using samples drawn by neonatal heel prick), the disease may present clinically with seizures, albinism (excessively fair hair and skin), and a “musty odor” to the baby’s sweat and urine (due to phenylacetate, one of the ketones produced). In most cases, a repeat test should be done at approximately two weeks of age to verify the initial test and uncover any phenylketonuria that was initially missed.

Untreated children are normal at birth, but fail to attain early developmental milestones, develop microcephaly, and demonstrate progressive impairment of cerebral function. Hyperactivity, EEG abnormalities, and seizures, and severe learning disabilities are major clinical problems later in life. A “musty or mousy” odor of skin, hair, sweat and urine (due to phenylacetate accumulation), as well as a tendency towards hypopigmentation and eczema, are also observed.

In contrast, affected children who are detected and treated are less likely to develop neurological problems or have seizures and mental retardation, though such clinical disorders are still possible

Classical PKU is caused by a mutated gene for the enzyme phenylalanine hydroxylase (PAH), which converts the amino acid phenylalanine to other essential compounds in the body. Other non-PAH mutations can also cause PKU. This is an example of non-allelic genetic heterogeneity. The PAH gene is located on chromosome 12 in the bands 12q22-q24.1. More than 400 disease-causing mutations have been found in the PAH gene. PAH deficiency causes a spectrum of disorders, including classic phenylketonuria (PKU) and hyperphenylalaninemia (a less severe accumulation of phenylalanine).

PKU is known to be an autosomal recessive genetic disorder. This means both parents must have at least one mutated allele of the PAH gene. The child must inherit both mutated alleles, one from each parent. Therefore, it is possible for a parent with the disease to have a child without it if the other parent possesses one functional allele of the gene for PAH. Yet, a child from two parents with PKU will inherit two mutated alleles every time, and therefore the disease.

Phenylketonuria can exist in mice, which have been extensively used in experiments into finding an effective treatment for it. The macaque monkey’s genome was recently sequenced, and the gene encoding phenylalanine hydroxylase was found to have the same sequence that, in humans, would be considered the PKU mutation.

 

Tetrahydrobiopterin-deficient hyperphenylalaninemia

 A rarer form of hyperphenylalaninemia occurs when PAH is normal, but there is a defect in the biosynthesis or recycling of the cofactor tetrahydrobiopterin (BH₄) by the patient. This cofactor is necessary for proper activity of the enzyme . The coenzyme (called biopterin) can be supplemented as treatment. Those who suffer from PKU must be supplemented with tyrosine to account for phenylalanine hydroxylase deficiency in converting phenylalanine to tyrosine sufficiently. Dihydrobiopterin reductase activity is to replenish quinonoid-dihydrobiopterin back into its tetrahydrobiopterin form, which is an important cofactor in many metabolic reactions in amino acid metabolism. Those with this deficiency may produce sufficient levels of phenylalanine hydroxylase, but since tetrahydrobiopterin is a cofactor for phenylalanine hydroxylase activity, deficient dihydrobiopterin reductase renders any phenylalanine hydroxylase enzyme produced unable to use phenylalanine to produce tyrosine. Tetrahydrobiopterin is also a cofactor in the production of L-DOPA from tyrosine and 5-Hydroxy-L-Tryptophan from tryptophan, which must also be supplemented as treatment in addition to the supplements for classical PKU.

Levels of dopamine can be used to distinguish between these two types. Tetrahydrobiopterin is required to convert phenylalanine to tyrosine, but it is also required to convert tyrosine to L-DOPA(via the enzyme tyrosine hydroxylase), which in turn is converted to dopamine. Low levels of dopamine lead to high levels of prolactin. By contrast, in classical PKU, prolactin levels would be relatively normal. Tetrahydrobiopterin deficiency can be caused by defects in four different genes. These types are known as HPABH4A, HPABH4B, HPABH4C, and HPABH4D.

The enzyme phenylalanine hydroxylase normally converts the amino acid phenylalanine into the amino acid tyrosine. If this reaction does not take place, phenylalanine accumulates and tyrosine is deficient. Excessive phenylalanine can be metabolized into phenylketones through the minor route, a transaminase pathway with glutamate. Metabolites include phenylacetate, phenylpyruvateand phenethylamine. Elevated levels of phenylalanine in the blood and detection of phenylketones in the urine is diagnostic, however most patients are diagnosed via newborn screening.

 Phenylalanine is a large, neutral amino acid (LNAA). LNAAs compete for transport across the blood–brain barrier (BBB) via the large neutral amino acid transporter (LNAAT). If phenylalanine is in excess in the blood, it will saturate the transporter. Excessive levels of phenylalanine tend to decrease the levels of other LNAAs in the brain. However, as these amino acids are necessary for protein and neurotransmitter synthesis, Phe buildup hinders the development of the brain, causing mental retardation.

If PKU is diagnosed early enough, an affected newborn can grow up with normal brain development, but only by managing and controlling Phe levels through diet, or a combination of diet and medication. Optimal health ranges (or “target ranges”) are between 120 and 360 µmol/L, and aimed to be achieved during at least the first 10 years. When Phe cannot be metabolized by the body, abnormally high levels accumulate in the blood and are toxic to the brain. When left untreated, complications of PKU include severe mental retardation, brain function abnormalities, microcephaly, mood disorders, irregular motor functioning, and behavioral problems such as attention deficit hyperactivity disorder.

All PKU patients must adhere to a special diet low in Phe for optimal brain development. “Diet for life” has become the standard recommended by most experts. The diet requires severely restricting or eliminating foods high in Phe, such as meat, chicken, fish, eggs, nuts, cheese, legumes, milk and other dairy products. Starchy foods, such as potatoes, bread, pasta, and corn, must be monitored. Infants may still be breastfed to provide all of the benefits of breastmilk, but the quantity must also be monitored and supplementation for missing nutrients will be required. The sweetener aspartame, present in many diet foods and soft drinks, must also be avoided, as aspartame contains phenylalanine.

Supplementary infant formulas are used in these patients to provide the amino acids and other necessary nutrients that would otherwise be lacking in a low-phenylalanine diet. As the child grows up these can be replaced with pills, formulas, and specially formulated foods. (Since Phe is necessary for the synthesis of many proteins, it is required for appropriate growth, but levels must be strictly controlled in PKU patients.) In addition, tyrosine, which is normally derived from phenylalanine, must be supplemented.

The oral administration of tetrahydrobiopterin (or BH4) (a cofactor for the oxidation of phenylalanine) can reduce blood levels of this amino acid in certain patients. The company BioMarin Pharmaceutical has produced a tablet preparation of the compound sapropterin dihydrochloride (Kuvan), which is a form of tetrahydrobiopterin. Kuvan is the first drug that can help BH4-responsive PKU patients (defined among clinicians as about 1/2 of the PKU population) lower Phe levels to recommended ranges. Working closely with a dietitian, some PKU patients who respond to Kuvan may also be able to increase the amount of natural protein they can eat.^[23] After extensive clinical trials, Kuvan has been approved by the FDA for use in PKU therapy. Some researchers and clinicians working with PKU are finding Kuvan a safe and effective addition to dietary treatment and beneficial to patients with PKU.

Several other therapies are currently under investigation, including gene therapy, large neutral amino acids, and enzyme substitution therapy with phenylalanine ammonia lyase (PAL). In the past, PKU-affected people were allowed to go off diet after approximately eight, then 18 years of age. Today, most physicians recommend PKU patients must manage their Phe levels throughout life.

For women with phenylketonuria, it is essential for the health of their children to maintain low Phe levels before and during pregnancy. Though the developing fetus may only be a carrier of the PKU gene, the intrauterine environment can have very high levels of phenylalanine, which can cross the placenta. The child may develop congenital heart disease, growth retardation, microcephaly and mental retardation as a result. PKU-affected women themselves are not at risk of additional complications during pregnancy.

In most countries, women with PKU who wish to have children are advised to lower their blood Phe levels (typically to between 2 and 6 mg/dL) before they become pregnant, and carefully control their levels throughout the pregnancy. This is achieved by performing regular blood tests and adhering very strictly to a diet, in general monitored on a day-to-day basis by a specialist metabolic dietitian. In many cases, as the fetus’ liver begins to develop and produce PAH normally, the mother’s blood Phe levels will drop, requiring an increased intake to remain within the safe range of 2–6 mg/dL. The mother’s daily Phe intake may double or even triple by the end of the pregnancy, as a result. When maternal blood Phe levels fall below 2 mg/dL, anecdotal reports indicate that the mothers may suffer adverse effects, including headaches, nausea, hair loss, and general malaise. When low phenylalanine levels are maintained for the duration of pregnancy, there are no elevated levels of risk of birth defects compared with a baby born to a non-PKU mother. Babies with PKU may drink breast milk, while also taking their special metabolic formula. Some research has indicated an exclusive diet of breast milk for PKU babies may alter the effects of the deficiency, though during breastfeeding the mother must maintain a strict diet to keep her Phe levels low. More research is needed. US scientist announced in June 2010 that they would be conducting a thorough investigation on the mutation of genes in the human genome. Their top priority is PKU, as it has become increasingly common, and sufferers often bear children who will be carriers of the recessive gene, and may themselves live past the age of sixty.

 

Testing for Genetic Disorders

Clinical Diagnosis.There is a large number of multiple congenital anomaly (MCA) syndromes whose diagnosis is based on gestalt — a clinical impression that quickly comes to mind on seeing the patient. The diagnosis of achondroplasia, for instance, rests solely on the clinical examination that may include anthropometric measurements and X-rays. There are no biochemical tests needed for confirmation.

Screening Tests. When available, laboratory tests can be invaluable in establishing a diagnosis or identifying individuals who carry genes coding for adult onset dominant or recessive genetic conditions. Screening tests for specific genetic disorders have as their primary goal early diagnosis, which makes timely treatment or prevention possible.

Newborn screening for PKU is a well-known example of a screening test. Phenylketonuria has an incidence of 1 in 10,000 to 1 in 15,000. If untreated, 95% of affected individuals will develop moderate to severe mental retardation. With dietary restriction of phenylalanine, virtually all patients with PKU will be mentally normal. The Guthrie test (bacterial inhibition assay) is one of the tests used to determine the phenylalanine level in the blood. Blood on filter paper is placed on agar plates with a strain of bacillus subtilis that requires phenylalanine for growth. The presence of growth is indicated by a halo surrounding the filter paper. If positive, blood phenylalanine and tyrosine levels are determined, and if elevated, a confirmatory assay for phenylalanine hydroxylase is done. Since phenylalanine is an essential amino acid (not produced by the body and comes from dietary sources), it is essential for a baby to be fed prior to testing. Ideally this test should be done on the second or third day of life. It is important to realize that this test is a screening test done to identify elevated phenylalanine levels. It is not diagnostic, as hyper-phenylalaninemia can be reversible, transient, or secondary to an elevated level of tyrosine, or other variants unrelated to classic PKU. In PKU the phenylalanine level is usually 20-40 mg/dl in comparison with normal levels of 4-6 mg/dl. On the average, 1 in 20 babies with positive screens will have classic PKU. PKU is one of the single gene disorders that can be diagnosed based on the presence of an elevated precursor protein or substrate.

Newborn screening programs are usually sponsored by the state and include tests for such conditions as PKU (Guthrie test), galactosemia (transferase assay), congenital hypothyroidism (T4, TSH determination), and hemoglobinopathies such as sickle cell anemia (isoelectric focusing or DNA diagnosis).

Prenatal screening, specifically maternal serum alpha fetoprotein (MSAFP) screening or amniotic fluid chromosome analysis, seeks to identify fetal pathology. The level of MSAFP is elevated in neural tube defects (NTDs) and low in certain chromosomal disorders. Since around 95% of NTDs occur in families with a negative family history and since NTDs and chromosome anomalies are serious disorders, MSAFP screening is now considered standard of care. Alpha-fetoprotein is made in the fetal liver and finds its way through the placenta to the maternal circulation. MSAFP determination is done in mid trimester (15-20 weeks gestation).

Diagnostic tests help physicians revise disease probability for their patients. All tests should be ordered by the physician to answer a specific question. The 5 main reasons for a diagnostic test are as follows:

         Establish a diagnosis in symptomatic patients. For example, an ECG to diagnose ST-elevation myocardial infarction (STEMI) in patients with chest pain.

         Screen for disease in asymptomatic patients. For example, a prostate-specific antigen (PSA) test in men older than 50 years.

         Provide prognostic information in patients with established disease. For example, a CD4 count in patients with HIV.

         Monitor therapy by either benefits or side effects. For example, measuring the international normalized ratio (INR) in patients taking warfarin.

         A test may be performed to confirm that a person is free from a disease. For example, a pregnancy test to exclude the diagnosis of ectopic pregnancy.

The criterion (reference) standard test definitively decides either presence or absence of a disease. Examples of criterion standard tests include pathological specimens for malignancies and pulmonary angiography for pulmonary embolism. However, criterion standard tests routinely come with drawbacks; they are usually expensive, less widely available, more invasive, and riskier. These issues usually compel most physicians to choose other diagnostic tests as surrogates for their criterion standard test.

For example, venography, the criterion standard for vein thrombosis, is an invasive procedure with significant complications including renal failure, allergic reaction, and clot formation. These risks make venography less desirable than the alternative diagnostic test—venous duplex ultrasonography. The price most diagnostic tests pay for their ease of use compared with their criterion standard is a decrease in accuracy. How to account for this trade-off between diagnostic accuracy and patient acceptability is the subject of this article.

In order for patients to make informed decisions regarding diagnostic and screening options when there is more than one option, wheo option has a clear advantage, and when the risk-benefit profile may be valued differently, decision aids such as pamphlets, videos, or Web-based tools may be used.

A Cochrane review of decision aids for patients facing treatment or screening decisions found that the use of these aids improved knowledge of the options and helped patients have more accurate expectations of possible benefits and harms, reach choices that are more consistent with informed values, and participate in decision making with health practitioners.^[1] Smaller improvements were seen with the use of more detailed decision aids compared to simpler decision aids. The use of decision aids had no apparent adverse effects on health outcomes or satisfaction.

Screening tests are laboratory tests that help to identify people with increased risk for a condition or disease before they have symptoms or even realize they may be at risk so that preventive measures can be taken. They are an important part of preventive health care.

Screening tests help detect disease in its earliest and most treatable stages. Therefore, they are most valuable when they are used to screen for diseases that are both serious and treatable, so that there is a benefit to detecting the disease before symptoms begin.

They should be sensitive – that is, able to correctly identify those individuals who have a given disease. Many routine tests performed at regular health exams are screening tests. Cholesterol testing and Pap smears for women are examples. Newborns are screened for a variety of conditions at birth.

A positive screening test often requires further testing with a more specific test. This is important in order to correctly exclude those individuals who do not have the given disease or to confirm a diagnosis.

A diagnostic test may be used for screening purposes, but a diagnostic test is generally used to confirm a diagnosis in someone who has signs, symptoms, or other evidence of a particular disease.

\Carrier Tests. Carrier testing for a gene disorder has as its primary goal to establish the carrier status of an individual or a couple so that they can exercise reproductive choice. The methods utilized to test for the above conditions include: biochemical tests for Тау-Sachs disease (hexosaminidase A), and congenital adrenal hyperplasia (21 hydroxylase); DNA testing for Duchenne muscular dystrophy (direct DNA mutation analysis), and fragile X syndrome (Southern blot hybridization or PCR); and karyotype analysis for carriers of chromosome abnormalities.

Knowing the carrier status of one or both parents can be critical to reproductive choice. There are a number of options available to couples who carry abnormal genes or chromosomes, including adoption or choosing not to have children. Some couples may choose to have children and take the chance that they will be affected. Others may opt for prenatal diagnosis and pregnancy termination. Some couples may choose prenatal diagnosis so they can make arrangements to deliver at a hospital where immediate treatment is available. Artificial insemination with a normal donor egg or sperm is another option some ut-risk couples may choose.

Presymtomatic Disorders. A third type of genetic testing is presymptomatic diagnosis, which has as its primary goal to identify the presence of an abnormal gene in at risk, but otherwise healthy, adults. These individuals come to clinical attention because of a positive family history of a genetic disorder.

A classic example is Huntington disease (HD). HD is an autosomal dominant trait with an average age of onset between the fourth and fifth decades. The gene has been mapped to the short arm of chromosome 4pl6.3. The HD gene has also been cloned and the abnormality identified as an expansion of a trinucleotide repeat sequence. The sequence of bases, CAG, is repeated in tandem 11 to 34 times iormal individuals. Thirty-nine or more copies of CAG are present in HD individuals. Individuals at risk can seek presymptomatic diagnosis prior to starting a family.

Presymptomatic diagnosis is also available to families at risk for autosomal dominant breast cancer. Again, these individuals come to clinical attention because of a positive family history of breast and/or ovarian cancer. TheBRCAl breast cancer gene has been identified on chromosome 17q. The gene for BRCA2 is on chromosome 13q. Individuals who carry the abnormal gene have an 80-90% lifetime risk of developing breast/ovarian cancer. Individuals may seek presymptomatic testing so that those carrying the gene can be more vigilant with breast examination and mammography, or alternatively opt for prophylactic mastectomy or oophorectomy. The diagnosis of Huntington disease and breast cancer involves DNA studies.

Presymptomatic diagnosis and, to a lesser extent, carrier testing, have numerous psychological, social, ethical, legal, and financial repercussions and need to be approached with caution. For this reason, there is a national consensus protocol for presymptomatic diagnosis of Huntington disease. This is a multistep process requiring four to five clinic visits with involvement of geneticists, psychiatrists, neurologists, psychologists and social workers.

Finally, confirmatory diagnosis is provided to the individuals seeking consultation. This can involve chromosomal, biochemical, or molecular DNA studies, as indicated by the individual cases.

The National Society of Genetic Counselors (NSGC) officially defines genetic counseling as the understanding and adaptation to the medical, psychological and familial implications of genetic contributions to disease.This process integrates:

·  Interpretation of family and medical histories to assess the chance of disease occurrence or recurrence.

·  Education about inheritance, testing, management, prevention, resources

·  Counseling to promote informed choices and adaptation to the risk or condition.

A genetic counselor is an expert with a Master of Science degree in genetic counseling. In the United States they are certified by the American Board of Genetic Counseling. In Canada, genetic counselors are certified by the Canadian Association of Genetic Counsellors. Most enter the field from a variety of disciplines, including biology, genetics, nursing, psychology, public health and social work. Genetic counselors should be expert educators, skilled in translating the complex language of genomic medicine into terms that are easy to understand.

Genetic counselors work as members of a health care team and act as a patient advocate as well as a genetic resource to physicians. Genetic counselors provide information and support to families who have members with birth defects or genetic disorders, and to families who may be at risk for a variety of inherited conditions. They identify families at risk, investigate the problems present in the family, interpret information about the disorder, analyze inheritance patterns and risks of recurrence, and review available genetic testing options with the family.

Genetic counselors are present at high risk or specialty prenatal clinics that offer prenatal diagnosis, pediatric care centers, and adult genetic centers. Genetic counseling can occur before conception (i.e. when one or two of the parents are carriers of a certain trait) through to adulthood (for adult onset genetic conditions, such as Huntington’s disease or hereditary cancersyndromes).

Patients

Any person may seek out genetic counseling for a condition they may have inherited from their biological parents.

A woman, if pregnant, may be referred for genetic counseling if a risk is discovered through prenatal testing (screening or diagnosis). Some clients are notified of having a higher individual risk forchromosomal abnormalities or birth defects. Testing enables women and couples to make a decision as to whether or not to continue with their pregnancy, and helps provide information that can be used to prepare for the birth of a child with medical issues.

A person may also undergo genetic counseling after the birth of a child with a genetic condition. In these instances, the genetic counselor explains the condition to the patient along with recurrence risks in future children. In all cases of a positive family history for a condition, the genetic counselor can evaluate risks, recurrence and explain the condition itself.

The goals of genetic counseling are to increase understanding of genetic diseases, discuss disease management options, and explain the risks and benefits of testing.^[3] Counseling sessions focus on giving vital, unbiased information and non-directive assistance in the patient’s decision making process. Seymour Kessler, in 1979, first categorized sessions in five phases: an intake phase, an initial contact phase, the encounter phase, the summary phase, and a follow-up phase. The intake and follow-up phases occur outside of the actual counseling session. The initial contact phase is when the counselor and families meet and build rapport. The encounter phase includes dialogue between the counselor and the client about the nature of screening and diagnostic tests. The summary phase provides all the options and decisions available for the next step. If counselees wish to go ahead with testing, an appointment is organized and the genetic counselor acts as the person to communicate the results.

Families or individuals may choose to attend counseling or undergo prenatal testing for a number of reasons.

·  Family history of a genetic condition or chromosome abnormality

·  Molecular test for single gene disorder

·  Increased maternal age (35 years and older)

·  Increased paternal age (40 years and older)

·  Abnormal maternal serum screening results or ultrasound findings

·  Increased nuchal translucency measurements on ultrasound

·  Strong family history of cancer

·  Predictive testing for adult-onset conditions

Detectable conditions

Many disorders cannot occur unless both the mother and father pass on their genes, such as Cystic Fibrosis. Some diseases can be inherited from one parent, such as Huntington disease, andDiGeorge syndrome. Other genetic disorders are the cause of an error or mutation occurring during the cell division process (e.g.trisomy). Testing can reveal conditions that are easily treatable as long as they are detected (Phenylketonuria or PKU). Results from genetic testing may also reveal:

·  Down syndrome

·  Sickle-cell anemia

·  Tay-Sachs disease

·  Spina bifida

·  Muscular dystrophy

·  Mental retardation

Each individual considers their family needs, social setting, cultural background, and religious beliefs when interpreting their risk. Clients must evaluate their reasoning to continue with testing at all. Counselors are present to put all the possibilities in perspective and encourage clients to take time to think about their decision. When a risk is found, counselors frequently reassure parents that they were not responsible for the result. An informed choice without pressure or coercion is made when all relevant information has been given and understood.

Prenatal genetic counseling

If an initial noninvasive screening test reveals a risk to the baby, clients are encouraged to attend genetic counseling to learn about their options. Further prenatal investigation is beneficial and provides helpful details regarding the status of the fetus, contributing to the decision-making process. Decisions made by clients are affected by factors including timing, accuracy of information provided by tests, and risk and benefits of the tests. Counselors present a summary of all the options available. Clients may accept the risk and have no future testing, proceed to diagnostic testing, or take further screening tests to refine the risk. Invasive diagnostic tests possess a small risk of miscarriage (1-2%) but provide more definitive results. While families seek direction and suggestions from the counselors, they are reassured that no right or wrong answer exists. When discussing possible choices, counselor discourse predominates and is characterized by examples of what some people might do. Discussion enables people to place the information and circumstances into the context of their own lives. Clients are given a decision-making framework they can use to situate themselves. Counselors focus on the importance of individual choice based on the experiences, morals, and viewpoints of the couple/individual/family. Testing is offered to provide a definitive answer regarding the presence of a certain genetic condition or chromosomal abnormality. There is ofteo therapy or treatment available for these conditions, and as such parents may have to make decisions regarding the management of the pregnancy.