Tuesday, July 29, 2008

Basic Genetic

Introduction to Medical Genetics
Current Medical Diagnosis & Treatment 2008

Physicians at one time concerned themselves only with what they could discover by bedside evaluation and laboratory investigation. In the parlance of genetics, the patient's symptoms and signs constitute his or her phenotype. Now the means are at hand for defining a person's genotype, the actual information content inscribed in the 2 m of coiled DNA present in each cell of the body—or half that amount in every mature ovum or sperm. Most phenotypic characteristics—and this includes diseases as well as human traits such as personality, adult height, and intelligence—are to some extent determined by the genes. The importance of the genetic contribution varies widely among human phenotypes, and methods are only now being developed to identify the genes involved in complex traits and most common diseases. Moreover, the importance of interactions between environment and genotype in producing phenotypes cannot be overstated despite the obscurity of the actual mechanisms.

The billions of nucleotides in the nucleus of a cell are organized linearly along the DNA double helix in functional units called genes, and each of the 25,000 or so human genes is accompanied by various regulatory elements that control when it is active in producing messenger RNA (mRNA) by a process called transcription. In most situations, mRNA is transported from the nucleus to the cytoplasm, where its genetic information is translated into proteins, which perform the functions that ultimately determine phenotype. For example, proteins serve as enzymes that facilitate metabolism and cell synthesis; as DNA binding elements that regulate transcription of other genes; as structural elements of cells and the extracellular matrix; and as receptor molecules for intracellular and intercellular communication.

Chromosomes are the vehicles in which the genes are carried from generation to generation. Each chromosome is a complex of protein and nucleic acid in which an unbroken double helix of DNA is coiled and supercoiled into a space many orders of magnitude less than the extended length of the DNA. Within the chromosome there occur highly complicated and integrated processes, including DNA replication, recombination, and transcription. In the nucleus of each somatic cell, humans normally have 46 chromosomes, which are arranged in 23 pairs. One of these pairs, the sex chromosomes X and Y, determines the sex of the individual; females have the pair XX and males the pair XY. The remaining 22 pairs are called autosomes (Figure 1). In addition to these nuclear chromosomes, each mitochondrion—found in varying numbers in the cytoplasm of all cells—contains multiple copies of a small chromosome. This mitochondrial chromosome encodes a few of the proteins for oxidative metabolism and all of the transfer RNAs used in translation of proteins within this organelle. Mitochondrial chromosomes are inherited almost entirely from the cytoplasm of the fertilized ovum and are therefore maternal in origin.

Fig. 1. Normal karyotype of a human male. Prepared from cultured amniotic cells and stained with Giemsa stain. About 400 bands are detectable per haploid set of chromosomes.

In all somatic cells, the 44 autosomes and one of the X chromosomes are transcriptionally active. In males, the active X is the only X; portions of the Y chromosome are also active. In females, the requirement for dosage compensation (to be equivalent to the situation in males) is satisfied by inactivation of most of one X chromosome early in embryogenesis. This process of X chromosomal inactivation, although incompletely understood, is known to be random, so that on average, in 50% of a female's cells, one of the X chromosomes will be active, and in the other 50% the homologous member of the pair will be active. The phenotype of the cell is determined by which genes on the chromosomes are active in producing mRNA at any given time.

Genes & Chromosomes

In all genes, information is contained in parcels called exons, which are interspersed with stretches of DNA called introns that do not encode any information about the protein sequence. However, introns may contain genetic regulatory sequences, and some introns are so large that they encode an entirely distinct gene.

The exact location of a gene on a chromosome is its locus, and the array of loci constitutes the human gene map. Currently, the chromosomal sites of more than 11,000 genes (for which normal or abnormal function has been identified) are known, often to a high degree of resolution. A variation of this map, identifying selected loci known to be involved in human disease, is shown in Figure 2. The difference in the higher resolution of the ordering of genes achievable by molecular techniques (such as linkage analysis) compared to cytogenetic techniques (such as visualization of small defects) is substantial, though the gap is narrowing. The chromosomes in the "standard" karyotype shown in Figure 1 have about 450 visible bands; under the best of cytologic and microscopic conditions, a total of about 1600 bands can be seen. But even in this extended configuration, each band contains dozens—sometimes hundreds—of individual genes. Thus, loss (deletion) of a small band will involve loss of many coding sequences and will have diverse effects on the phenotype.

Fig.2. A partial "morbid map" of the human genome. Shown next to the ideogram of the human X and Y chromosomes are representative mendelian disorders caused by mutations at that locus. Over 460 phenotypes have been mapped to the X chromosome and 8 to the Y chromosome.

The number and arrangement of genes on homologous chromosomes are identical even though the actual coding sequences of homologous genes may not be. Homologous copies of a gene are termed alleles. In comparing alleles, it must be specified at what level of analysis the comparison is being made. When alleles are truly identical—in that their coding sequences are invariant—the individual is homozygous at that locus. At a coarser level, the alleles may be functionally identical despite subtle variations in nucleotide sequence—with the result either that the proteins produced from the two alleles are identical or that whatever differences there may be in amino acid sequence will have no bearing on the function of the protein. If the individual is being analyzed at the level of the protein phenotype, allelic homozygosity would again be an apt descriptor. However, if the analysis were at the level of the DNA—as occurs in restriction enzyme examination or nucleotide sequencing—then, despite functional identity, the alleles would be viewed as different and the individual would be heterozygous for that locus. Heterozygosity based on differences in the protein products of alleles has been detectable for decades and was the first hard evidence concerning the high degree of human biologic variability. In the past decade, analysis of DNA sequences has shown this variability to be much more remarkable—differences in nucleotide sequence between individuals occur about once every 400 nucleotides.


Allelic heterozygosity most often results when different alleles are inherited from the egg and the sperm, but it also occurs as a consequence of spontaneous alteration in nucleotide sequence (mutation). Genetic change occurring during formation of an egg or a sperm is called a germinal mutation. When the change occurs after conception—from the earliest stages of embryogenesis to dividing cells in the body of the oldest adult—it is termed a somatic mutation. As is discussed below, the role of somatic mutation in the etiology of human disease is now increasingly recognized.

The coarsest type of mutation is alteration in the number or physical structure of chromosomes. For example, nondisjunction (failure of chromosome pairs to separate) during meiosis—the reduction division that leads to production of mature ova and sperms—causes the embryo to have too many or too few chromosomes, a situation called aneuploidy. Rearrangement of chromosome arms, such as occurs in translocation or inversion, is a mutation even if breakage and reunion do not disrupt any coding sequence. Thus, the phenotypic effect of gross chromosomal mutations can range from profound (as in aneuploidy) to nil.

A bit less coarse, but still detectable cytologically, are deletions of part of a chromosome. Such mutations almost always alter phenotype, because a number of genes are lost; however, a deletion may involve only a single nucleotide, whereas about 1–2 million nucleotides (1–2 megabases) must be lost before the defect can be visualized by the most sensitive cytogenetic methods short of in situ hybridization. Molecular biologic techniques are needed to detect smaller losses.

Mutations of one or a few nucleotides in exons have several potential consequences. Changes in one nucleotide can alter which amino acid is encoded; if the amino acid is in a critical region of the protein, function might in this way be severely disturbed (eg, sickle cell disease). On the other hand, some amino acid substitutions have no detectable effect on function, and the phenotype is therefore unaltered by the mutation. Similarly, because the genetic code is degenerate (two or more different three-nucleotide sequences called codons encode the same amino acids), nucleotide substitution does not necessarily alter the amino acid sequence of the protein. Three specific codons signal termination of translation; thus, a nucleotide substitution in an exon that generates one of the stop codons usually causes a truncated protein, which is nearly always dysfunctional. Other nucleotide substitutions can disrupt the signals that direct splicing of the mRNA molecule and grossly alter the protein product. Finally, insertions and deletions of one or more nucleotides can have dramatic effects—any change that is not a multiple of three nucleotides disrupts the reading frame of the remainder of the exon—or potentially minimal effects (if the protein can tolerate the insertion or loss of an amino acid).

Mutations in introns may disrupt mRNA splicing signals or may be entirely silent with respect to the phenotype. A great deal of variation in nucleotide sequences among individuals (averaging one difference every few hundred nucleotides) resides within introns. Mutations in the DNA between adjacent genes may also be silent or may have a profound effect on phenotype if regulatory sequences are disrupted. A novel mechanism for mutation, which also helps explain clinical variation among relatives, has been discovered in myotonic dystrophy, Huntington disease, fragile X mental retardation syndrome, Friedreich ataxia, and other disorders. A region of repeated trinucleotide sequences close to or within a gene can be unstable in some families; expansion of the number of repeated units within this segment beyond a critical threshold is associated with a more severe phenotype, a phenomenon termed anticipation.

Mutations may occur spontaneously or may be induced by environmental factors such as radiation, medication, or viral infections. Both advanced maternal and paternal age favor mutation, but of different types. In women, meiosis is completed only when an egg ovulates, and chromosomal nondisjunction is increasingly common as the egg becomes older. The risk that an aneuploid egg will result increases exponentially and becomes a major clinical concern for women older than their early 30s. In men, mutations of a subtler sort—affecting nucleotide sequences—increase with age. Offspring of men over age 40 years are at an increased risk for having mendelian conditions, primarily autosomal dominant ones.

Genes in Individuals

For some quantitative traits such as adult height or serum glucose concentration in normal individuals, it is difficult to distinguish the contributions of individual genes; this is because in general, phenotypes are the products of multiple genes acting in concert. However, if one of the genes in the system is aberrant, a major departure from the "normal" or expected phenotype might arise. Whether the aberrant phenotype is serious (ie, a disease) or even recognized depends on the nature of the defective gene product and how resilient the system is to disruption. The latter point emphasizes the importance of homeostasis in both physiology and development—many mutations go unrecognized because the system can cope, even though tolerances for further perturbation might be narrowed.

In other words, most human characteristics are polygenic, whereas many of the disordered phenotypes thought of as "genetic" are monogenic but still influenced by other loci in a person's genome.

Phenotypes due to alterations at a single gene are also characterized as mendelian, after Gregor Mendel, the monk and part-time biologist who studied the reproducibility and recurrence of variation in garden peas. He showed that some traits were dominant to other traits, which he called recessive. The dominant traits required only one copy of a "factor" to be expressed, regardless of what the other copy was, whereas the recessive traits required two copies before expression occurred. In modern terms, the mendelian factors are genes, and the alternative copies of the gene are alleles. Let A be the common (normal) allele and let a be a mutant allele at a locus: If the same phenotype is present whether the genotype is A/a or a/a, the phenotype is dominant, whereas if the phenotype is present only when the genotype is a/a, it is recessive.

In medicine, it is important to keep two considerations in mind: First, dominance and recessiveness are attributes of the phenotype, not the gene; and second, the concepts of dominance and recessiveness depend on how the phenotype is defined. To illustrate both points, consider sickle cell disease. This condition occurs when a person inherits two alleles for βS-globin, in which the normal glutamate at position 6 of the protein has been replaced by valine; the genotype for the β-globin locus is HbS/HbS, compared to the normal HbA/HbA. When the genotype is HbS/HbA, the individual does not have sickle cell disease, so this condition satisfies the criteria for being a recessive phenotype. But now consider the phenotype of sickled erythrocytes. Red cells with the genotype HbS/HbS clearly sickle—but, if the oxygen tension is reduced, so do cells with the genotype HbS/HbA. Therefore, sickling is a dominant trait.

A mendelian phenotype is characterized not only in terms of dominance and recessiveness but also according to whether the determining gene is on the X chromosome or on one of the 22 pairs of autosomes. Traits or diseases are therefore called autosomal dominant, autosomal recessive, X-linked recessive, and X-linked dominant.

Genes in Families

Since the first decade of the twentieth century, the patterns of recurrence of specific human phenotypes have been explained in terms of principles first described by Mendel in the garden pea plant. Mendel's second principle—usually referred to as his first1—is called the law of segregation and states that a pair of factors (alleles) that determines some trait separates (segregates) during formation of gametes. In simple terms, a heterozygous (A/a) person will produce two types of gametes with respect to this locus—one containing only A and one containing only a, in equal proportions. Offspring of this person will have a 50–50 chance of inheriting the A allele and a similar chance of inheriting the a allele.

1 Mendel's first law stated that—from the perspective of the phenotype—it mattered not from which parent a particular mutant allele was inherited. For years this principle was thought to be too obvious to be codified as anybody's "law" and was therefore ignored. In fact, however, recent evidence from studies of human disorders suggests that certain genes are "processed" (imprinted) as they move through the gonad and that processing in the testis is different from that in the ovary. Thus, not only is this first mendelian principle important, it was incorrect as originally formulated from observations in peas.

The concepts of genes in individuals and in families can be combined to specify how mendelian traits will be inherited.

Autosomal Dominant Inheritance

The characteristics of autosomal dominant inheritance in humans can be summarized as follows: (1) There is a vertical pattern in the pedigree, with multiple generations affected (Figure 3). (2) Heterozygotes for the mutant allele show an abnormal phenotype. (3) Males and females are affected with equal frequency and severity. (4) Only one parent must be affected for an offspring to be at risk for developing the phenotype. (5) When an affected person mates with an unaffected one, each offspring has a 50% chance of inheriting the affected phenotype. This is true regardless of the sex of the affected parent—specifically, male-to-male transmission occurs. (6) The frequency of sporadic cases is positively associated with the severity of the phenotype. More precisely, the greater the reproductive fitness of affected persons, the less likely it is that any given case resulted from a new mutation. (7) The average age of fathers is advanced for isolated (sporadic or new mutation) cases.

Fig.3. A pedigree illustrating autosomal dominant inheritance. Square symbols indicate males and circles females; open symbols indicate that the person is phenotypically unaffected, and filled symbols indicate that the phenotype is present to some extent.

Autosomal dominant phenotypes are often age dependent, less severe than autosomal recessive ones, and associated with malformations or other physical features. They are pleiotropic in that multiple, even seemingly unrelated clinical manifestations derive from the same mutation, and variable in that expression of the same mutation among people will differ.

Penetrance is a concept associated with mendelian conditions—especially dominant ones—and the term is often misused. It should be defined as an expression of the frequency of appearance of a phenotype (dominant or recessive) when one or more mutant alleles are present. For individuals, penetrance is an all-or-none phenomenon—the phenotype is either present (penetrant) or not (nonpenetrant). The term variability—not "incomplete penetrance"—should be used to denote differences in expression of an allele.

The most frequent cause of apparent nonpenetrance is insensitivity of the methods for detecting the phenotype. If an apparently normal parent of a child with a dominant condition was in fact heterozygous for the mutation, the parent would have a 50% chance at each subsequent conception of having another affected child. A common cause of nonpenetrance in adult-onset mendelian diseases is death of the affected person before the phenotype becomes evident but after transmission of the mutant allele to offspring. Thus, accurate genetic counseling demands careful attention to the family medical history and high-resolution scrutiny of both parents of a child with a condition known to be a mendelian dominant trait.

When both alleles are expressed in the heterozygote, as in blood group AB, in sickle trait (HbS/HbA), in the major histocompatibility antigens (eg, A2B5/A3B17), or in sickle-C disease (HbS/HbC), the phenotype is called codominant.

In human dominant phenotypes, the mutant allele in homozygotes is almost always more severe than in heterozygotes.

Autosomal Recessive Inheritance

The characteristics of autosomal recessive inheritance in humans can be summarized as follows: (1) There is a horizontal pattern in the pedigree, with a single generation affected (Figure 4). (2) Males and females are affected with equal frequency and severity. (3) Inheritance is from both parents, each a heterozygote (carrier) and each usually clinically unaffected. (4) Each offspring of two carriers has a 25% chance of being affected, a 50% chance of being a carrier, and a 25% chance of inheriting neither mutant allele. Thus, two-thirds of all clinically unaffected offspring are carriers. (5) In matings between individuals, each with the same recessive phenotype, all offspring will be affected. (6) Affected individuals who mate with unaffected individuals who are not carriers have only unaffected offspring. (7) The rarer the recessive phenotype, the more likely it is that the parents are consanguineous (related).

Fig. 4. A pedigree illustrating autosomal recessive inheritance. (Symbols as in Figure 3.)

Autosomal recessive phenotypes are often associated with deficient activity of enzymes and are thus termed inborn errors of metabolism. Such disorders include phenylketonuria, Tay-Sachs disease, and the various glycogen storage diseases and tend to be more severe, less variable, and less age dependent than dominant conditions.

When an autosomal recessive condition is quite rare, the chance that the parents of affected offspring are consanguineous is increased. As a result, the prevalence of rare recessive conditions is high among inbred groups such as the Old Order Amish. On the other hand, when the autosomal recessive condition is common, the chance of consanguinity between parents of cases is no higher than in the general population (about 0.5%).

Two different mutant alleles at the same locus, as in HbS/HbC, form a genetic compound (compound heterozygote). The phenotype usually lies between those produced by either allele present in the homozygous state. Because of the large number of mutations possible in a given gene, many autosomal recessive phenotypes are probably due to genetic compounds. Sickle cell disease is an exception. Consanguinity is strong presumptive evidence for true homozygosity of mutant alleles and against a genetic compound.

X-Linked Inheritance

The general characteristics of X-linked inheritance in humans can be summarized as follows: (1) There is no male-to-male transmission of the phenotype (Figure 5). (2) Unaffected males do not transmit the phenotype. (3) All of the daughters of an affected male are heterozygous carriers. (4) Males are usually more severely affected than females. (5) Whether a heterozygous female is counted as affected—and whether the phenotype is called "recessive" or "dominant"—depends often on the sensitivity of the assay or examination. (6) Some mothers of affected males will not themselves be heterozygotes (ie, they will be homozygous normal) but will have a germinal mutation. The proportion of heterozygous (carrier) mothers is negatively associated with the severity of the condition. (7) Heterozygous women transmit the mutant gene to 50% of their sons, who are affected, and to 50% of their daughters, who are heterozygotes. (8) If an affected male mates with a heterozygous female, 50% of the male offspring will be affected, giving the false impression of male-to-male transmission. Of the female offspring of such matings, 50% will be affected as severely as the average hemizygous male; in small pedigrees, this pattern may simulate autosomal dominant inheritance.

Fig.5. A pedigree illustrating X-linked inheritance. (Symbols as in Figure 3.)

The characteristics of X-linked inheritance depend on phenotypic severity. For some disorders, affected males do not survive to reproduce. In such cases, about two-thirds of affected males have a carrier mother; in the remaining third, the disorder arises by new germinal mutation in an X chromosome of the mother. When the disorder is nearly always manifest in heterozygous females (X-linked dominant inheritance), females tend to be affected about twice as often as males; and on average an affected female transmits the phenotype to 50% of her sons and 50% of her daughters.

X-linked phenotypes are often clinically variable—particularly in heterozygous females—and suspected of being autosomal dominant with nonpenetrance. For example, Fabry disease (α-galactosidase A deficiency) may be clinically silent in carrier women or may cause stroke, renal failure, or myocardial infarction by middle age.

Germinal mosaicism occurs in mothers of boys with X-linked conditions. The chance of such a mother having a second affected son or a heterozygous daughter depends on the fraction of her oocytes that carries the mutation. Currently, this fraction is impossible to determine. However, the presence of germinal mosaicism can be detected in some conditions (eg, Duchenne muscular dystrophy) in a family by analysis of DNA, and this knowledge becomes crucial for genetic counseling.

Mitochondrial Inheritance

Mutations in the genes encoded by the mitochondrial chromosome cause a variety of diseases that affect (in particular) organs highly dependent on oxidative metabolism, such as the retina, brain, kidneys, and heart. Because a person's mitochondria derive almost entirely from the ovum, the inheritance pattern is distinct from that of mendelian disorders and is termed "maternal" or, more appropriately, "mitochondrial." An affected woman can pass the defective mitochondrial chromosome to all of her offspring, whereas an affected man has little risk of passing his mutation to a child (Figure 6). Because each cell and the ovum contain many mitochondria and because each mitochondrion contains many chromosomes, two situations are possible: If every chromosome in every mitochondrion carries the same mutation, the person is said to be homoplasmic for the mutation. On the other hand, if only some of the mitochondrial chromosomes carry the mutation, the person is heteroplasmic. In the latter case, an offspring may inherit relatively few mitochondria bearing the mutation and have mild disease or no disease.

Fig. 6. Mitochondrial ("maternal") inheritance. A mitochondrial genetic mutation, indicated by darkened symbols, is passed by the female (circle) to all of her offspring, including males (squares). Of subsequent offspring, males do not transmit the mutation, but females continue to transmit the mutation to all of their offspring because mitochondria are passed through ova, not sperm. For simplicity, although both parents are shown for the first generation, subsequent generations do not show the genetic partners, who are assumed to lack the mutation. Note: All or only some of the mitochondria may carry the mutation, a variable that affects the clinical expression of the mutation. (See text regarding homoplasmic and heteroplasmic individuals.)

Over 16,000 human genes have been identified or implied through their phenotypes and inheritance patterns in families. This total represents 60–70% of all genes thought to be encoded by the 22 autosomes, two sex chromosomes, and the mitochondrial chromosome. Victor McKusick and colleagues coordinate an international effort to catalogue human mendelian variation.

Disorders of Multifactorial Causation

Many disorders cluster in families but are not associated with evident chromosomal aberrations or mendelian inheritance patterns. Examples include congenital malformations such as cleft lip, pyloric stenosis, and spina bifida; coronary artery disease; type 2 diabetes mellitus; and various forms of neoplasia. They are often characterized by varying frequencies in different racial or ethnic groups, disparity in sexual predilection, and greater frequency (but less than full concordance) in monozygotic than in dizygotic twins. This inheritance pattern is called "multifactorial" to signify that multiple genes interact with various environmental agents to produce the phenotype. The familial clustering is assumed to be due to sharing of both alleles and environment.

For most multifactorial conditions, there is little understanding of which particular genes are involved, how they and their products interact, and in what way different nongenetic factors contribute to the phenotype. For some disorders, biochemical and genetic studies have identified mendelian conditions within the coarse phenotype: Defects of the low-density lipoprotein receptor account for a small fraction of cases of ischemic heart disease (a larger fraction if only patients under age 50 years are considered); familial polyposis of the colon predisposes to adenocarcinoma; and some patients with emphysema have inherited deficiency of α1-proteinase inhibitor (α1-antitrypsin). Despite these notable examples, this reductionistic preoccupation with mendelian phenotypes is unlikely to explain the great majority of human disease; but even so, in the last analysis, much of human pathology will prove to be associated with genetic factors in cause, pathogenesis, or both.

Our ignorance about fundamental genetic mechanisms in development and physiology has not completely restricted practical approaches to the genetics of multifactorial disorders. For example, recurrence risks are based on empirical data derived from observation of many families. The risk of recurrence of multifactorial disorders is increased in several instances: (1) in close relatives (siblings, offspring, and parents) of an affected individual; (2) when two or more members of a family have the same condition; (3) when the first case in a family is in the less commonly affected sex (eg, pyloric stenosis is five times more common in boys; an affected woman has a threefold to fourfold greater risk of having a child with pyloric stenosis); and (4) in ethnic groups in which there is a high incidence of a particular condition (eg, spina bifida is 40 times more common in whites—and even more frequent among the Irish—than in Asians).

For many apparently multifactorial disorders, too few families have been examined to have established empirical risk data. A useful approximation of recurrence risk in close relatives is the square root of the incidence. For example, many common congenital malformations have an incidence of 1:2500 to 1:400 live births; the calculated recurrence risks are thus in the 2–5% range—values that correspond closely to experience.

Chromosomal Aberrations

Any deviation from the structure and number of chromosomes as displayed in Figure 1 is, technically, a chromosomal aberration. Not all aberrations cause problems in the affected individual, but some that do not may lead to problems in offspring. About 1:200 live-born infants has a chromosomal aberration that is detected because of some effect on phenotype. This frequency increases markedly the earlier in fetal life the chromosomes are examined. By the end of the first trimester of gestation, most fetuses with abnormal numbers of chromosomes have been lost through spontaneous abortion. For example, Turner syndrome—due to the absence of one sex chromosome and the presence of a single X chromosome—is a relatively common condition, but it is estimated that only 2% of fetuses with this form of aneuploidy survive to term. Even more striking in live-born children is the complete absence of most autosomal trisomies and monosomies despite their frequent occurrence in young fetuses.

Types of Chromosomal Abnormalities

Major structural changes occur in either balanced or unbalanced form. In the latter, there is a gain or loss of genetic material; in the former, there is no change in the amount of genetic material but only a rearrangement of it. At the sites of breaks and new attachments of chromosome fragments, there may be permanent structural or functional damage to one gene or to only a few genes. Despite no visible loss of material, the aberration may nonetheless be recognized as unbalanced through an abnormal phenotype and the chromosomal defect confirmed by molecular analysis of the DNA.

Aneuploidy results from nondisjunction—the failure of a chromatid pair to separate in a dividing cell. Nondisjunction in either the first or second division of meiosis results in gametes with abnormal chromosomal constitutions. In aneuploidy, more or fewer than 46 chromosomes are present (Table 1). The following are all forms of aneuploidy: (1) monosomy, in which only one member of a pair of chromosomes is present; (2) trisomy, in which three chromosomes are present instead of two; and (3) polysomy, in which one chromosome is represented four or more times.

Table 1. Clinical phenotypes resulting from aneuploidy.



Incidence at Birth

Trisomy 13

47,XX or XY,+13


Trisomy 18

47,XX or XY,+18


Trisomy 21 (Down syndrome)

47,XX or XY,+21


Klinefelter syndrome


1:600 males

XYY syndrome


1:1000 males

Turner syndrome


1:2500 females

XXX syndrome


1:1200 females

If nondisjunction occurs in mitosis, mosaic patterns occur in somatic tissue, with some cells having one karyotype and other cells of the same organism another karyotype. Patients with a mosaic genetic constitution often have manifestations of each of the genetic syndromes associated with the various abnormal karyotypes.

Translocation results from an exchange of parts of two chromosomes.

Deletion is loss of chromosomal material.

Duplication is the presence of two or more copies of the same region of a given chromosome. The redundancy may occur in the same chromosome or in a nonhomologous chromosome. In the latter case, a translocation will also have occurred.

An isochromosome is a chromosome in which the arms on either side of the centromere have the same genetic material in the same order—ie, the chromosome has at some time divided in such a way that it has a double dose of one arm and absence of the other.

In an inversion, a chromosomal region becomes reoriented 180 degrees out of the ordinary phase. The same genetic material is present, but in a different order.

The Techniques of Medical Genetics

Hereditary disorders affect multiple organ systems and people of all ages. Many disorders are chronic ones, although acute crises often occur. The concerns of patients and families span a wide range of medical, psychological, social, and economic issues. These characteristics emphasize the need for pediatricians, internists, obstetricians, and family practitioners to provide medical genetics services for their patients. This section reviews the laboratory and consultative services available from clinical geneticists and the indications for their use.

Family History & Pedigree Analysis

The first step in considering how important genetic factors might be in the clinical situation of a patient is obtaining a detailed family history. At a minimum, a patient should be queried in detail about all first-degree relatives—parents, siblings, and offspring—(age, sex, health status if alive, including major illnesses; cause of death) and more distant relatives with reference to the particular condition at issue. Ethnicity of both sides of the family should be noted; any disorders known to be especially prevalent in a particular ethnic group should be asked about specifically. Once the family history is obtained, it should be analyzed; medical geneticists and genetic counselors are trained in this task and are particularly valuable when the busy clinician has neither the time nor the staff to pursue the information. A pedigree diagram (eg, Figure 3) with the symbols filled in to indicate the presence of a condition can be instructive in suggesting a mode of inheritance. Once targeted genetic testing of the proband produces a result, the diagram also proves useful in identifying relatives who might benefit from counseling about similar testing.


Cytogenetics is the study of chromosomes by light microscopy. The chromosomal constitution of a single cell or an entire individual is specified by a standardized notation. The total chromosome count is determined first, followed by the sex chromosome complement and then by any abnormalities. The autosomes are all designated by numbers from 1 to 22. A plus (+) or minus (–) sign indicates, respectively, a gain or loss of chromosomal material. For example, a normal male is 46,XY, whereas a girl with Down syndrome caused by trisomy 21 is 47,XX,+21; a boy with Down syndrome caused by translocation of chromosome 21 to chromosome 14 in a sperm or an egg is 46,XY,–14,+t(14;21).

Chromosomal analyses are done by growing human cells in tissue culture, chemically inhibiting mitosis, and then staining, observing, photographing, sorting, and counting the chromosomes. The display of all of the chromosomes is termed the karyotype (Figure 1) and is the end result of the technical aspect of cytogenetics.

Specimens for cytogenetic analysis can be obtained for routine analysis from the peripheral blood, in which case T lymphocytes are examined; from amniotic fluid for culture of amniocytes; from trophoblastic cells from the chorionic villus; from bone marrow; and from cultured fibroblasts, usually obtained from a skin biopsy. Enough cells must be examined so that the chance of missing a cytogenetically distinct cell line (a situation of mosaicism) is statistically low. For most clinical indications, 20 mitoses are examined and counted under direct microscopic visualization, and two are photographed and karyotypes prepared. Observation of aberrations usually prompts more extended scrutiny and in many cases further analysis of the original culture.

A variety of methods can be used to reveal banding patterns—unique to each pair of chromosomes—in the analysis of aberrations. The number of bands that can be visualized is a function of how "extended" the chromosomes are, which in turn depends primarily on how early in metaphase (or even in prophase for the most extensive banding) mitosis was arrested. The "standard" karyotype reveals about 400 bands per haploid set of chromosomes, whereas a prophase karyotype might reveal four times that number. As invaluable as extended karyotypes are in certain clinical circumstances, their interpretation is often difficult—in terms of the time and effort required and of ambiguity about what is abnormal, what is a normal variation, and what is a technical artifact. In situ hybridization with DNA probes for specific chromosomes or regions of chromosomes can be labeled and used to identify subtle aberrations. Given proper technique, fluorescent in situ hybridization (FISH) yields sensitivities and specificities of almost 100%. Some applications are being used routinely and marketed commercially, though the Food and Drug Administration (FDA) has only recently begun to approve probes for clinical use.

An area of particular relevance is the use of FISH to detect heretofore undetectable deletions in the regions of chromosomes just proximal to their tips (subtelomeric). A remarkable number of patients with unexplained mental retardation or dysmorphology have been found to have such deletions, either because of de novo mutation or because of rearrangements due to balanced parental translocations. Comparative genomic hybridization (CGH) provides a method, using DNA chip-based technology, to survey a patient's genome for many more cytogenetically undetectable deletions than current FISH approaches. The chips in current commercial use contain 400–600 probes that include all sites of deletions and insertions of known clinical importance as well as regions of uncertain significance. On the immediate horizon are systems capable of scanning many thousands of regions of the human genome.

Indications for Cytogenetic Analysis

The current indications are listed in Table 2. A wide array of clinical syndromes has been found to be associated with chromosomal aberrations, and analysis of the karyotype is useful any time a patient is discovered to have the manifestations of one of these syndromes. When a chromosomal aberration is revealed, not only does the patient's physician obtain valuable information about prognosis, but the parents gain insight into the cause of their child's problems and the family can be counseled accurately—and usually reassured—about the risks of recurrence.

Table 2. Indications for cytogenetic analysis.

- Patients of any age who are grossly retarded physically or mentally, especially if there are associated anomalies.

- Any patient with ambiguous internal or external genitalia or suspected hermaphroditism.

- Girls with primary amenorrhea and boys with delayed pubertal development. Up to 25% of patients with primary amenorrhea have a chromosomal abnormality.

- Males with learning or behavioral disorders who are taller than expected (based on parental height).

- Certain malignant and premalignant diseases (see Tables 44–7 and 44–8).

- Parents of a patient with chromosome translocation.

- Parents of a patient with a suspected chromosomal syndrome if there is a family history of similarly affected children.

- Couples with a history of multiple spontaneous abortions of unknown cause.

- Couples who are infertile after more common obstetric and urologic causes have been excluded.

- Prenatal diagnosis (see Table 6).

Mental retardation is a frequent component of congenital malformation syndromes. Any person with unexplained mental retardation should be studied by chromosomal analysis.

The tips of chromosomes are called telomers. Recent studies using fluorescent probes for the genetic sequences just proximal to the telomers have shown subtle rearrangements or deletions in about 6% of patients who have otherwise unexplained mental retardation with or without dysmorphic features. These chromosomal aberrations are generally too small to be detected by routine cytogenetic analysis.

Abnormalities of sexual differentiation can be understood only after the patient's genetic sex is clarified. Hormonal therapy and plastic surgery can to some extent determine phenotypic sex, but genetic sex is dictated by the complement of sex chromosomes. The best-known example of dichotomy between the genetic sex and phenotypic sex is the testicular feminization syndrome, in which the chromosomal constitution is 46,XY but, because of a defect in the testosterone receptor protein (specified by a gene on the Y chromosome), the external phenotype is completely female.

Failure or delay in developing secondary sexual characteristics occurs in Turner syndrome (the most common cause being a form of aneuploidy, ie, monosomy for the X chromosome, 45,X), in Klinefelter syndrome (the most common karyotype is 47,XXY), and in other much rarer chromosomal aberrations.

Tall stature is perhaps the only consistent phenotypic feature associated with having an extra Y chromosome (karyotype 47,XYY); most men with this chromosomal aberration lead normal lives, and thus tall stature in a male is itself no indication for chromosomal analysis. However, some evidence suggests that an increased prevalence of learning difficulties may be associated with this aberration. Furthermore, Klinefelter syndrome often causes tall stature, albeit with a eunuchoid habitus, and learning and behavioral problems. Thus, the combination of learning or behavioral difficulties and unexpectedly increased height in a male should prompt consideration of cytogenetic analysis.

As discussed below, most tumors are associated with chromosomal aberrations, some of which are highly specific for certain malignancies. Cytogenetic analysis of tumor tissue may assist in diagnosis, prognosis, and management.

Whenever a person is shown to have a chromosome translocation—whether it be balanced and asymptomatic or unbalanced, causing a syndrome—the physician should consider the importance of identifying the source of the translocation. If the proband is a child and the parents are interested in having more children, both parents should be studied cytogenetically. How far the primary physician or consultant should go in tracking a translocation through a family is an unsettled question with legal and ethical as well as medical implications. Certainly the proband (if an adult) or the parents of the proband need to be counseled and the potential risks to relatives discussed. The physician should document, both in the medical record and by correspondence, that the burden of disclosing relevant data to the extended family has been assumed by specific named individuals.

Inability to produce offspring, either through failure to conceive or as a result of repeated miscarriages, is a frustrating and discouraging problem for affected couples and their physicians. Considerable progress in the urologic and gynecologic understanding of infertility has benefited many couples. However, chromosomal aberrations remain an important problem in reproductive medicine, and cytogenetic analysis should be utilized at some stage in extended evaluation. Infertility is common in both Klinefelter and Turner syndromes, either of which may be associated with mild external signs—particularly if the chromosomal aberration is mosaic. Any early spontaneous abortion may be due to fetal aneuploidy. Recurrence may be due to parental translocation predisposing to an unbalanced fetal karyotype.

Biochemical Genetics

Biochemical genetics deals not only with enzymatic defects but also with proteins of all functions, including cytoskeletal and extracellular structure, regulation, and receptors. The principal functions of the biochemical genetics laboratory are to determine the presence or absence of proteins, to assess the qualitative characteristics of proteins, and to verify the effectiveness of proteins in vitro. The key elements from the referring clinician's perspective are: (1) to indicate what the suspected clinical diagnoses are and (2) to make certain that the proper specimen is obtained and transported to the laboratory in a timely manner.

Indications for Biochemical Investigations

Some inborn errors are relatively common in the general population, eg, hemochromatosis, defects of the low-density lipoprotein receptor, and cystic fibrosis (Table 3). Others, although rare across the entire population, are common in certain ethnic groups, such as Tay-Sachs disease in Ashkenazic Jews, sickle cell disease in African Americans, and thalassemias in populations from around the Mediterranean basin and Asia. Many of these disorders are autosomal recessive, and the frequency of heterozygotes is many times that of the fully expressed disease. Screening for carrier status can be effective if certain requirements are satisfied (Table 4). For example, all of the United States and the District of Columbia require screening of newborns for phenylketonuria and often other metabolic diseases. Such programs are cost-effective even for rare conditions such as phenylketonuria, which occurs in only one of every 11,000 births. Unfortunately, not all disorders that meet the requirements in Table 4 are screened for in every state. Furthermore, compliance is highly variable among programs, and follow-up diagnostic tests, management, and counseling are in some cases inadequate. Babies most likely to be missed are those born at home and those discharged before they have digested much milk or formula. In some states, parents can refuse to have their infants studied. Several commercial laboratories have marketed screening for over 35 inborn errors of metabolism to hospitals. This supplemental newborn screening involves tandem mass spectroscopic analysis of the same blood spots used in state-mandated programs.

Table 3. Representative inborn errors of metabolism.

General Class of Defect


Biochemical Defect




Phenylalanine hydroxylase


Connective tissue

Osteogenesis imperfecta type II

α1(I) and α2(I) procollagen



Tay-Sachs disease

Hexosaminidase A


Glycogen storage disease

Type I



Immune function

Chronic granulomatous disease

Cytochrome b, β-chain


Lipid metabolism

Familial hypercholesterolemia

Low-density lipoprotein receptor


Mucopolysaccharidosis (MPS)

MPS II (Hunter's syndrome)

Iduronate sulfatase



Acute intermittent

Porphobilinogen deaminase



Cystic fibrosis (CF)

CF transmembrane conductance regulator


Urea cycle


Arginosuccinate synthetase


AR, autosomal recessive; AD, autosomal dominant; XL, X-linked recessive.

Table 4. Requirements for effective population screening for inborn errors of metabolism.

1. The disease should be clinically severe or have potentially severe consequences.

2. The natural history of the disease should be understood.

3. Effective treatment should be generally available and depend on early diagnosis for optimal results.

4. The disease incidence should be high enough to warrant screening.

5. The screening test should have favorable specificity (low false-positive rate) and sensitivity (low false-negative rate).

6. The screening test should be available for and used by the entire population at risk.

7. An adequate system for follow-up of positive results should be provided.

8. The economic cost-benefit analysis should favor screening and treatment.

Use of the biochemical genetics laboratory for other than screening purposes must be justified by the need for data on which to base a diagnosis of specific disorders or classes of related disorders. The possibilities are limited only by the extent of knowledge, the enthusiasm of the primary clinician or consultant, the willingness of the patient or family to pursue the diagnosis and specimens to be taken, and the availability of a laboratory to examine the specimens.

Though many inborn defects are so subtle they escape detection, there are a number of clinical situations in which an inborn error should be part of the differential diagnosis. The urgency with which the investigation is undertaken will vary depending on the severity of the disorder and the availability of treatment. Table 5 lists various clinical presentations.

Table 5. Manner of presentation of inborn errors of metabolism.

Presentation and Course


Acute metabolic disease of the neonate

Galactosemia, urea cycle disorders

Chronic disorders with little progression after infancy

Phenylketonuria, hypothyroidism

Chronic disorders with insidious, incessant progression

Tay-Sachs disease

Disorders causing abnormalities of structure

Skeletal dysplasias, Marfan's syndrome

Disorders of transport

Cystinuria, lactase deficiency

Disorders that determine susceptibilities

Low-density lipoprotein receptor deficiency, agammaglobulinemia

Episodic disorders

Most porphyrias, glucose-6-phosphate deficiency

Disorders causing anemia

Pyruvate kinase deficiency, hereditary spherocytosis

Disorders interfering with hemostasis

Hemophilia A and B, von Willebrand disease

Congenital disorders with no possibility of reversal

Testicular feminization

Disorders with protean manifestations

Pseudohypoparathyroidism, hereditary amyloidoses

Inborn errors with no clinical effects

Pentosuria, histidinemia

The possibility of acute metabolic disease of the neonate is the most important indication, because prompt diagnosis and treatment may often make the difference between life and death. The clinical features are nonspecific because the newborn has a limited repertoire of responses to severe metabolic insults. The physician must be both inclusive and systematic in evaluating such ill babies.

DNA Analysis

Direct inspection of nucleic acids—often called "molecular genetics" or "DNA diagnosis"—is achieving an increasingly prominent role in a number of clinical areas, including oncology, infectious disease, forensics, and the general study of pathophysiology. A major impact has been in the diagnosis of mendelian disorders. Molecular testing is available for more than 1000 separate hereditary conditions. Once a particular gene is shown to be defective in a given condition, the nature of the mutation itself can be determined, often by sequencing the nucleotides and comparing the array with that of a normal allele. One of a variety of techniques can then be used to determine whether that same mutation is present in other patients with the same disorder. Genetic heterogeneity is so extensive that most mendelian conditions are associated with numerous mutations at one locus—or occasionally multiple loci—that produce the same phenotype. Mutations at no less than 35 different genes cause nonsyndromic retinitis pigmentosa, and changes in at least 12 genes cause familial hypertrophic cardiomyopathy. This fact complicates DNA diagnosis of patients and screening for carriers of defects in specific genes.

A few conditions are associated with relatively few mutations or with only one highly prevalent mutation. For example, all sickle cell disease is caused by exactly the same change of glutamate to valine at position 6 of β-globin, and that substitution in turn is due to a change of one nucleotide at the sixth codon in the β-globin gene. But such uniformity is the exception. In cystic fibrosis, about 70% of white heterozygotes have an identical deletion of three nucleotides that causes loss of a phenylalanine residue from a chloride transport protein; however, the remaining 30% of mutations of that protein are diverse (over 1200 have been discovered), so that no simple screening test will detect all carriers of cystic fibrosis.

Reviews of the current technical status of DNA analysis appear regularly in the medical literature. Polymerase chain reaction (PCR) studies have revolutionized many aspects of molecular biology, and DNA diagnosis has come to involve this technique in many instances. If the sequences of the 10–20 nucleotides at the ends of a region of DNA of interest (such as a portion of a gene) are known, then "primers" complementary to these sequences can be synthesized. When even a minute amount of DNA from a patient (eg, from a few leukocytes, buccal mucosal cells, or hair bulbs) is combined with the primers in a reaction mixture that replicates DNA—and after several dozen cycles are then performed—the region of DNA between the primers will be amplified exponentially. For example, the presence of early HIV infection can be detected after PCR amplification of a portion of the viral genome. A variety of new techniques are being studied in an attempt to permit analysis of many potential genetic variations in one individual in a very short time. For example, "DNA chip arrays," about the size of a microscope cover slip, can contain tens of thousands of specific nucleotide sequences. When an individual's DNA is denatured and allowed to hybridize to the array, pairing of sequences causes a fluorescent signal that can be detected by a laser microscope and recorded and interpreted by a computer.

Indications for DNA Diagnosis

The basic requirement for the use of nucleic acids in the diagnosis of hereditary conditions is that a probe be available for the gene in question. The probe may be a piece of the actual gene, a sequence close to the gene, or just a few nucleotides at the actual mutation. The closer the probe is to the actual mutation, the more accurate and the more useful will be the information derived. DNA diagnosis involves one of two general approaches: (1) direct detection of the mutation or (2) linkage analysis, whereby the presence of a mutation is inferred from the nature of a probe DNA sequence remote from the mutation. In the latter approach, as the probe moves farther from the mutation, the chances increase that recombination will have separated the two sequences and confused the interpretation of the data.

DNA diagnosis is finding frequent application in presymptomatic detection of individuals with age-dependent disorders such as Huntington disease and adult polycystic kidney disease, screening for carriers of autosomal recessive conditions such as cystic fibrosis and thalassemias, screening for female heterozygotes of X-linked conditions such as Duchenne muscular dystrophy and hemophilia A and B, and prenatal diagnosis (see below). For some conditions in which serious complications occur in adolescence or early adulthood—such as von Hippel–Lindau syndrome, hereditary hemorrhagic telangiectasia, and familial polyposis coli—genetic testing at an early age can identify those relatives who need frequent clinical monitoring and prophylactic management; almost as important, relatives who test negative for the mutation can be spared the inconvenience, cost, and risk of clinical tests. In all instances of DNA testing, primary care providers and specialists alike must be mindful that substantive ethical, psychological, legal, and social issues remain unresolved. For example, some conditions for which hereditary susceptibility can be readily defined (such as Alzheimer disease, Huntington disease, and many cancers) have no effective therapy at this time. For these same conditions, health insurance and life insurance providers may be especially interested in learning who among their current or prospective customers is at higher risk. Some states have enacted legislation to protect people identified as having a heightened genetic risk of disease.

Logistics of DNA Diagnosis

Lymphocytes are a ready source of DNA; 10 mL of whole blood yields up to 0.5 mg of DNA, enough for dozens of analyses based on hybridization, each of which requires only 5 mcg. If the analysis is quite narrowly focused on a specific mutation (such as in a family study, in which only one specific nucleotide change is addressed), PCR analysis can often be used and the amount of DNA needed is truly infinitesimal—a few hair bulbs or sperm are adequate. Once isolated, the DNA sample can be divided into aliquots and frozen. Alternatively, lymphocytes can be transformed with viruses into lymphoblasts; these cells are immortal, can be frozen, and—whenever DNA is required—can be thawed, propagated, and their DNA isolated. These stored specimens provide access to a person's genome long after the individual dies. This is such an important advantage that many clinical genetics centers and commercial laboratories "bank" DNA from patients and informative relatives even if the samples cannot be put to use immediately. The specimens may later prove invaluable to relatives or to other patients being evaluated. DNA in some instances has become more reliable than the medical records.

Blood for DNA isolation should be drawn in ethylenediaminetetraacetic acid (EDTA) anticoagulant (lavender-top tubes); blood for lymphoblast culture should be drawn in heparin (green-top tubes). Neither should be frozen. Specimens for DNA isolation can be stored or shipped at room temperature over a period of a few days. Lymphoblast cultures should be established within 48 hours, so prompt shipment is essential. For one or a few specific DNA analyses, some laboratories accept a swab that has been placed between the cheek and gum for a minute (buccal swab); enough cells adhere to the fibers that DNA from the subject can be isolated.

Fetal DNA can be isolated from amniotic cells, from trophoblastic cells taken by chorionic villus sampling, or from either cell type grown in culture. Samples need to be processed promptly but can be shipped by overnight mail and must not be frozen.

Prenatal Diagnosis

It is possible to diagnose in utero, before the middle of the second trimester, several hundred mendelian disorders, all chromosome aberrations, and a number of congenital malformations that are not mendelian. The first step toward prenatal diagnosis is taken when the expecting couple, the primary care provider, or the obstetrician thinks of the need for it. Recent surveys suggest that even for the most common indication for such service—advanced maternal age—less than 50% of all women 35 years and older in the United States are offered prenatal testing.

Techniques Used in Prenatal Diagnosis

Prenatal diagnosis depends on the ability to assay the fetus directly (fetal blood sampling, fetoscopy), indirectly (analysis of amniotic fluid, amniocytes or trophoblastic cells, ultrasound), or remotely (analysis of maternal serum). Some of these techniques satisfy the requirements for screening (Table 4) and should be offered to all pregnant women; others carry considerable risk and should be reserved for specific circumstances. A few centers are developing preimplantation diagnosis of the embryo; a single cell is plucked from the six- to eight-cell blastocyst, which has been cultured after in vitro fertilization, generally without harming future development. The chromosomes of the cell can be studied by FISH or the genes by PCR. Another new approach, with considerable potential, is isolation of fetal cells that are circulating in minute numbers in the maternal circulation.

Ultrasound scanning of the fetus is a safe, noninvasive procedure that can diagnose gross skeletal malformations as well as nonbony malformations known to be associated with specific diseases. Some obstetricians routinely perform fetal ultrasound at least once between 12 and 20 weeks of gestation.

Other prenatal diagnostic procedures—fetoscopy, fetography, and amniography—are more invasive and a definite risk to the mother and fetus. They are indicated only if the risk of the suspected abnormality is high and the information cannot be obtained by other means.

All of the cytogenetic, biochemical, and DNA analytic techniques discussed above can be applied to specimens from the fetus. Aside from screening for α-fetoprotein in maternal serum to detect neural tube defects, analysis of fetal chromosomes is the most frequently performed test. Chromosomal analysis can be performed on amniotic cells and on trophoblastic cells grown in culture and directly on any trophoblastic cells that happen to be undergoing mitosis. Amniotic fluid cells are derived primarily from the fetal urinary system. Amniocentesis can be performed during gestational weeks 16–18 to permit unhurried sample analysis, transmission of results, and reproductive decisions. The time from obtaining the sample to a final reading of the karyotype has now been shortened to a week or so, and automated methods may reduce the time a bit further. Chorionic villus sampling (CVS) for trophoblastic cells (derived embryologically from the same fertilized egg as the fetus) is usually done during gestational weeks 10–13. If the tissue can be analyzed directly, cytogenetic results can be obtained within a few hours; however, the quality of the karyotypes is inferior to that from cultured cells, and most laboratories routinely culture cells and reexamine any suspected abnormalities. The advantage of CVS is that the results are available early in pregnancy, so that termination, if elected, can occur earlier in the pregnancy and the obstetric complications of termination are fewer.

The risk of CVS is somewhat higher than that of amniocentesis, though both are relatively safe. Between 0.5% and 1% of pregnancies are lost as a complication of CVS, whereas less than one in 300 amniocenteses result in fetal loss. Some centers offer "early amniocentesis," performed during gestational weeks 12–14; the magnitude of the risks is similar to that of CVS. These figures are lower than—but are in addition to—the 2–3% spontaneous abortion rate after the first trimester ends.

Indications for Prenatal Diagnosis

The indications for prenatal diagnosis are listed in Table 6. A few deserve comment.

Table 6. Indications for prenatal diagnosis.



Advanced maternal age, one parent a carrier of a translocation, previous child with chromosome aberration, intrauterine growth delay

Cytogenetics (amniocentesis, chorionic villus sampling)

Biochemical disorder

Protein assay, DNA diagnosis

Congenital anomaly

Ultrasound, fetal cytogenetics

Screening for neural tube defects and trisomy

Maternal multiple marker screening, ultrasound

Most studies done for advanced maternal age will detect no chromosomal aberration, and the couple will be reassured by this news. However, it is always appropriate to emphasize that the average risk of producing a child with a defect evident at birth, such as a physical malformation or some inborn error of metabolism, is about 3%, and that the risk increases with the age of either parent. Simply examining the chromosomes reduces this risk minimally. On the other hand, unless one of the other indications is present, it is simply not possible to "screen" a pregnancy for most birth defects (neural tube defects being an exception).

Individuals contemplating pregnancy—especially those of Ashkenazic Jewish or other white ethnicity—should be offered screening for the most common mutations in the CFTR gene that cause cystic fibrosis. If both partners are detected as being carriers, prenatal diagnosis of a fetus would be an option for them.

A history for cytogenetic aberrations emphasizes a chromosomal defect in a parent, a family history of a chromosomal defect, or a previous child or conceptus with a defined or undefined chromosomal defect. The factors that render some couples susceptible to repeated episodes of aneuploidy are unclear, and routine prenatal testing is warranted once a defect has occurred.

Cytogenetic analysis of the fetus will of course provide information about the sex chromosomes. Some couples do not desire advance knowledge of the sex of their child, and the person transmitting the results to the couple should always address this issue first. On the other hand, some couples only want to know the sex of the fetus and plan to terminate the pregnancy if the undesired sex is detected. Few centers in the United States consider sex selection to be an appropriate indication for prenatal diagnosis.

The level of α-fetoprotein in maternal serum changes with gestational age, with the mother's medical status, and with abnormalities of the fetus. If the first two factors can be well controlled, the assay can be used to provide information about the fetus. Levels are expressed as multiples of the median value for a particular gestational age. Higher than normal levels are associated with open neural tube defects (the conditions for which the test was developed), recent or impending fetal demise, gastroschisis, and fetal renal disease. Extremely high levels are highly specific for fetal anomalies—a level three times the median increases 20-fold the risk of meningomyelocele or anencephaly. Low α-fetoprotein levels in maternal serum are associated with fetal trisomy, especially Down syndrome; the reason for this association remains unclear. The addition of two other analytes in maternal serum—human chorionic gonadotropin (hCG) and unconjugated estriol (uE3)—to the assay for α-fetoprotein (to produce the "triple screen") enhances by several times the ability to detect a fetus with trisomy 21 and trisomy 18. Measuring in the first trimester both serum pregnancy-associated plasma protein A and the translucency of the fetal neck by ultrasound—followed by routine triple screening in the second trimester—improves the rate of detection of Down syndrome to about 85% while reducing false-positive results to about 1%. A positive result on any of these screening protocols for trisomy should be followed by offering the woman amniocentesis to confirm the diagnosis.

Neoplasia: Chromosomal & DNA Analysis

Studies of both chromosomes and nucleic acids support Boveri's 1914 hypothesis that cancer is caused by a change in genetic material at the cellular level. Two classes of genes have been discovered that function in neoplastic transformation.

Oncogenes arise from preexisting normal genes (protooncogenes) that have been altered by both viral and nonviral factors. As a result, the cells synthesize either normal proteins in inappropriate amounts or proteins that are aberrant in structure and function. Many of these proteins are cellular growth factors or receptors for growth factors. The net result of oncogene activation is unregulated cell division. Mutations that activate oncogenes almost always arise in somatic cells and are not usually inherited. Although some oncogenes are more likely to be activated in certain tumors, in general the same mutations may be found in neoplasia arising in different cells and tissues.

Tumor suppressor genes can be viewed as the antithesis of oncogenes. Their normal function is to suppress transformation; mutation in both alleles is necessary to obliterate this important function. The first mutant allele at any tumor suppressor gene might arise spontaneously or might be inherited; mutation in the other allele (the "second hit") almost always arises spontaneously, but by any of a number of molecular mechanisms. These genes show considerably more tumor specificity than do oncogenes; however, although some specific mutations are necessary for certain tumors to arise, no loss of single tumor suppressor function is sufficient. Clearly, a person who inherits one copy of a mutant tumor suppressor gene is at increased risk that in some susceptible cell, at some time during life, the function of that gene will be lost. This susceptibility is inherited as an autosomal dominant trait. For example, mutation in one allele of the p53 locus results in the Li-Fraumeni syndrome (151623), in which susceptibility before age 45 years to sarcomas and other tumors occurs in males and females in successive generations. Inherited mutations in this locus also increase the risk that a second tumor will develop following radiation or chemotherapy for the first tumor, suggesting that the initial treatment may induce a "second hit" in a p53 locus in another tissue. However, inheriting a p53 mutation is not a guarantee that cancer will develop at an early age; much more needs to be learned about the pathogenesis of neoplasia before the genetic counseling of families with a molecular predisposition to cancer is clarified. BRCA1, a gene that predisposes women to breast (114480) and ovarian cancer, is another example of a tumor suppressor gene. Women who inherit one mutant allele of BRCA1 have, on average, a 60–80% lifetime risk of developing breast cancer, and the average age of tumor detection is in the fifth decade. Their risk of developing ovarian cancer is 34–45%. For both females and males with certain mutations in BRCA1, the risks of colon and pancreatic cancer are increased severalfold over that of the general population.

In selected cases, a patient's DNA can be analyzed for the presence of a mutated gene and thereby that individual's risk for developing a tumor can be assessed. Examples are retinoblastoma (189200), certain forms of Wilms tumor (194070), breast cancer (114489), and familial colon cancer (114500). To illustrate how noninvasive and sensitive the methodology has become, it is possible to analyze stool for the presence of mutations in tumor suppressor genes that might indicate the presence of a clinically undetected adenocarcinoma of the colon. The analysis—not yet in general use—depends on the ability of the PCR to amplify minute quantities of the mutant DNA present in epithelial cells shed from the tumor.

A third class of genes that predispose to malignancy has been discovered in the past few years. So-called mutator genes have joined oncogenes and tumor suppressor genes as risk factors. Mutator genes normally function to repair damage to DNA that occurs from environmental insults such as exposure to carcinogens and ultraviolet irradiation. When a mutator gene is mutated itself, DNA damage accumulates and eventually affects oncogenes and tumor suppressor genes, thereby making cancer more likely. Hereditary nonpolyposis colon cancer (HNPCC) is one familial syndrome due to mutations in one or another of the five mutator genes identified thus far (MSH2 and MLH1 being the most commonly responsible for HNPCC).

This exciting work on the molecular nature of oncogenesis was preceded by years of study of the cytogenetics of tumors. Indeed, the retinoblastoma tumor suppressor gene was ultimately isolated because a small number of patients with this tumor have a constitutive deletion of chromosome 13 where this gene maps. Other chromosomal aberrations have been found to be highly characteristic of—or even specific for—certain tumors (Table 7). Detection of one of these cytogenetic aberrations can thus aid in diagnosis.

Table 7. Chromosome aberrations associated with representative solid tumors.


Chromosome Aberration




del(1)(p36), del(11)(q23)

Renal cell carcinoma

del(3)(p14.2–p25) or translocation of this region

Retinoblastoma, osteosarcoma

del(13)(q14.1) or translocation of this region

Small-cell lung carcinoma


Wilms' tumor


1Nomenclature means, "a deletion at band q11 of chromosome 22."

Hematologic malignancies are especially amenable to study because of the relative ease of performing cytogenetic analysis. Such malignancies are associated with over 100 specific chromosomal rearrangements, primarily translocations. Most of these rearrangements are restricted to a specific type of cancer (Table 8), and the remainder occur with many cancers.

Table 8. Chromosomal aberrations associated with representative hematologic malignancies.


Chromosomal Aberration


Acute myeloblastic


Acute promyelocytic


Acute monocytic


Chronic myelogenous





B cell


T cell

inv, del, and t of 1p13–p12


Polycythemia vera


1Nomenclature means, "a translocation with the union at band q22 of chromosome 8 and q11 of chromosome 21."

In the leukemias, the chromosomal aberration is the basis of one of the subclassifications of the disease. When cytogenetic information is combined with the histologic classification, it is possible to define subsets of patients in whom response to therapy, clinical course, and prognosis are predictable. If at the time of diagnosis there are no chromosomal changes in the bone marrow cells, the survival time is longer than if any or all of the bone marrow cells have abnormal cytogenetic characteristics. As secondary chromosomal changes occur, the leukemia becomes more aggressive, often associated with drug resistance and a reduced chance for complete or prolonged remission. The least ominous chromosomal change is numerical alteration without morphologic abnormality.

Less cytogenetic information is available for lymphomas and premalignant hematologic disorders than for leukemia. In Hodgkin's disease, studies have been limited by the low yield of dividing cells and the low number of clear-cut aneuploid clones, so that complete chromosomal analyses with banding are available for far fewer patients with Hodgkin's disease than for any other type of lymphoma. In Hodgkin disease, the modal chromosomal number tends to be triploid or tetraploid. About one-third of the samples have a 14q+ chromosome. In non-Hodgkin lymphomas, high-resolution techniques of banding detect abnormalities in 95% of cases. Cytogenetic findings are now being correlated with the immunologic and histologic features and with prognosis.

In Burkitt lymphoma, a solid tumor of B cell origin, 90% of patients have a translocation between the long arm of chromosome 8 and the long arm of chromosome 14, with chromosomal breakage sites being at or near immunoglobulin and oncogene loci.

Instability of chromosomes also predisposes to the development of some malignancies. In certain autosomal recessive diseases such as ataxia-telangiectasia, Bloom's syndrome, and Fanconi's anemia, the cells have a tendency to genetic instability, ie, to chromosomal breakage and rearrangement in vitro. These diseases are associated with a high incidence of neoplasia, particularly leukemia and lymphoma.

Some chromosomal aberrations, better known for their effect on phenotype, also predispose to tumors. For example, patients with Down syndrome (trisomy 21) have a 20-fold increase in the risk of leukemia, 47,XXY males (Klinefelter syndrome) have a 30-fold increase in the risk of breast cancer, and XY phenotypic females have a heightened risk of developing ovarian cancer, primarily gonadoblastoma.

The indications for cytogenetic analysis of neoplasia continue to evolve. Not all tumors require study. However, in cases of tumors of unclear type (especially leukemias and lymphomas), with a strong family history of early neoplasia, or for certain tumors associated with potential generalized chromosomal defects (present in nonneoplastic cells), cytogenetic analysis should be strongly considered.


Yudhi Gejali said...

saya ngelink juga ok,!

-Indonesian Medical