Genetic Variation

The human genome contains just over 3 billion base pairs and the sequence is about 99.9% identical in all individuals. It is a surprising finding that human genetic variability may be due to just 0.1% difference in the human genome sequence. A single-nucleotide polymorphism, or SNP, is the most common DNA variant in the genome, with about 10 million occurrences. SNPs are single-base substitutions and occur on average in 1 in every 1250 nucleotides. SNPs are polymorphisms in that a base substitution does not cause a change in the phenotype; they are found in more than 1% of the population. Because of their ubiquity in the human genome SNPs have become invaluable in identifying sequence changes associated with disease risk through traditional linkage studies and population-based association studies. Although not thought to alter protein coding, SNPs have been associated with diseases by their presence in regulatory control regions or introns. SNPs may also determine a disease’s response to treatment, as with the angiotensin-II type 1 receptor polymorphism and its association with congestive heart failure.⁸

Normal Karyotype

Our cells contain 23 pairs of chromosomes; 22 pairs are autosomes and shared between males and females, and one pair is the sex chromosomes, XX and XY. The ordered arrangement of chromosomes is a karyotype. Routine chromosome analysis involves drawing blood and stimulating lymphocytes in culture into rapid division. Cell division is then inhibited at metaphase and the chromosomes fixed on glass slides. The chromosomes are then stained and photographed for analysis.

Chromosome Structure

Chromosomes are composed of short arms, p, and long arms, q. The chromosome has a primary constriction, or centromere, where the microtubules attach for cell division. The telomeres, or tips of the chromosomes, are capped with a repeating sequence TTAGGG that is critical for the maintenance of chromosome integrity. The relative position of the centromere further delineates the structure of the chromosome. In humans the acrocentric chromosomes 13, 14, 15, 21, and 22 are characterized by stalks and satellites where genes for ribosomal RNA are located (Fig. 5-2).

Figure 5-2 Human chromosomes. These chromosomes represent the pattern seen with G banding or Giemsa staining. The different types of chromosomes and elements of chromosome structure are noted. The bands for chromosome 17 are numbered.

Numerical Abnormalities

Abnormalities of chromosome number are the most commonly recognized clinical chromosome abnormalities. Structural chromosome anomalies contribute significantly to birth defects, infertility, and recurrent pregnancy loss. Abnormalities in chromosome number generally arise from mistakes at cell division where there is either gain or loss, or both, of chromosomes in daughter cells, or nondisjunction. Nondisjunction can occur either during meiosis or mitosis. Cells resulting from this occurrence are aneuploid because their chromosome number is not a multiple of the haploid number 23. Nondisjunction can occur during either meiosis I or meiosis II. Either can produce an aneuploid conception (Fig. 5-3). Nondisjunction or anaphase lag can occur at mitosis, causing mosaicism, the presence of two or more cell lines in an individual. Mosaicism is often seen with the sex chromosome abnormality Turner’s syndrome, where up to 50% of cases have some form of mosaicism.⁹ The common numerical chromosome abnormalities are listed in Table 5-1. Numerical chromosome abnormalities can also involve multiples of the haploid number of 23 chromosomes. Triploidy, with 69 chromosomes, usually arises from the fertilization of a single egg by two sperm. Triploid conceptions are seen in about 15% of chromosomally abnormal miscarriages and occasionally survive to term. Tetraploidy has a modal number of 92 chromosomes and occurs in a small percentage of spontaneous losses.

Figure 5-3 Nondisjunction. A, Nondisjunction can occur at meiosis I, producing an aneuploid gamete and trisomic conception. The polar bodies are represented by empty circles. B, Postzygotic or mitotic nondisjunction can occur, producing somatic mosaicism. This is seen frequently in Turner’s syndrome. C, Nondisjunction in meiosis II can produce normal gametes, along with those with 22 and 24 chromosomes.

Table 5-1 Chromosome Abnormalities

Structural Chromosome Abnormalities

Structural chromosomal rearrangements occur when chromosomes break and the original architecture is not restored. Chromosome rearrangements are balanced when the diploid genetic state is maintained. When rearrangements are unbalanced, they result in aneuploidy for one or more chromosome segments. These structural rearrangements may segregate and be termed familial, or they may occur as a first or new event, de novo. Balanced familial chromosome rearrangements are in most cases truly balanced and represent little risk of birth defects or mental retardation. De novo chromosome rearrangements that appear balanced carry a small risk of aneuploidy at the molecular level of about 5% for birth defects and developmental delay.

Translocations involve the exchange of chromosome arms between two different chromosomes. Reciprocal translocations occur when there is breakage within two arms and reciprocal exchange of the distal segments, creating a derivative chromosome (Fig. 5-4). In most cases balanced translocation carriers are phenotypically normal but are at risk for producing unbalanced gametes during gametogenesis. In a balanced translocation carrier the types of chromosome segregation can be complex, resulting in a normal segregation pattern, a balanced translocation pattern, and an unbalanced pattern producing a partial trisomy and partial monosomy for the chromosomes involved (see Fig. 5-4).

Figure 5-4 Reciprocal translocation between two nonhomologous chromosomes. A, Germ line chromosomes of a balanced translocation carrier. B, Alternate segregation produces gametes with normal chromosomes and balanced translocation products. C, Adjacent-1 segregation produces unbalanced gametes, resulting in partial monosomy and partial trisomy conceptions.

When the short arms of two acrocentric chromosomes are involved in a translocation, the long arms are joined in the centromeric region of one chromosome, with the loss of the short arms of the acrocentric chromosomes producing Robertsonian translocations. Because the short arms of acrocentric chromosomes contain redundant ribosomal genetic material, the loss of this material is of no phenotypic consequence. As with balanced translocations, the products of meiotic segregation can be either balanced or unbalanced (Fig. 5-5).

Figure 5-5 Robertsonian translocation. A translocation has occurred between two acrocentric chromosomes. A, Normal and balanced segregants will produce genetically-balanced offspring. B, The unbalanced segregants will produce translocation Down syndrome and the nonviable monosomy 21.

Other structural abnormalities can produce pregnancy loss and birth defects. When two breaks occur in a single chromosome with the interstitial segment flipped 180 degrees at the time of repair, an inversion can occur. If this involves each arm of the chromosome a pericentric inversion is produced. If only a single arm of the chromosome is involved in the inversion, a paracentric inversion is produced (Fig. 5-6). Each has unique and different implications for gamete production and pregnancy loss. With chromosome duplication a chromosome segment of varying size can be duplicated, causing a partial trisomy for this segment. With a deletion, a segment of varying size is missing, causing a genetic imbalance or partial monosomy. This condition, in which a second copy of a gene or segment of chromosome is missing, resulting in an abnormal phenotype or clinical presentation, is known as haploinsufficiency.

Figure 5-6 The inversion on the left is a pericentric inversion involving the centromere. The inversion on the right is a paracentric inversion involving a single chromosome arm.

Fluorescent In Situ Hybridization

Traditional cytogenetics has always been limited by the band level or resolution of the karyotype. Even with high-resolution banding that could elongate chromosomes and resolve an increasing number of bands per chromosome, one could never be certain that a chromosome was intact at the molecular level. With advances in DNA technology and cytogenetics it is now possible using fluorescent in situ hybridization (FISH) to analyze chromosomes at the molecular level for changes in the DNA. Molecular cytogenetics has now revolutionized cytogenetics by permitting (1) the analysis of DNA structure within a chromosome down to within 10 to 100 kb and (2) the diagnostic analysis of nondividing interphase cells, producing a significant impact on the field of prenatal diagnosis and that of preimplantation genetic diagnosis.¹⁰

FISH technology uses DNA probes that can bind or anneal to specific DNA sequences within the chromosome. A denatured probe is incubated with native DNA from a cell that has also been denatured to the single-strand state. The probe substitutes biotin-dUTP or digoxigenin-UTP for thymidine. After the probe has annealed to native DNA, the probe–DNA complex can be detected by adding fluorochrome-tagged avidin that binds to biotin or fluorochrome-labeled antidigoxigenin. This signal can be additionally amplified by adding antiavidin and the complex visualized by fluorescence microscopy. Using several different fluorochromes tagged to different DNA probes, different chromosomes or chromosome segments can be simultaneously visualized within a cell as different colored signals. The ability to detect specific gene segments that are either present or missing has permitted the diagnosis of contiguous gene syndromes at the DNA level as well as translocations in interphase nuclei, often in single cells.

Material for FISH can be either metaphase chromosomes obtained from dividing cells or interphase nuclei from cells that are not dividing. Slides are pretreated with RNAse and proteinase to remove RNA that may cross-hybridize with the probe and chromatin. The slides are heated in formamide to denature the DNA and then fixed in cold ethanol. The probe is then prepared for hybridization by heating. The probe and chromosome preparation are then mixed and sealed with a coverslip at 37°C for hybridization. By varying the incubation temperature or the salt composition of the hybridization solution, the stringency of the binding can be increased and the background labeling reduced.

Applications of FISH

The technique of in situ hybridization first proved useful for localizing genes to chromosomes. With the introduction of fluorescence labeling, in situ hybridization proved invaluable in identifying chromosome abnormalities that could not be identified by traditional banding methods. FISH also played a key role in one of the most unusual discoveries of modern genetics, that of genomic imprinting.

FISH technology has been developed in three forms. Centromeric or alpha-satellite probes are relatively chromosome specific and have had probably the broadest application in interphase genetics.^11,12 These probes produce somewhat diffuse signals near the centromere with adequate strength, but do not cross-hybridize with chromosomes that have similar centromeric sequences. Single copy probes have now been developed that give discrete signals from a specific band on a chromosome and avoid the issue of cross-hybridization. These can also be used to detect copy number and specific chromosome regions known to be associated with syndromes. Single copy probes and centromeric probes for chromosomes 13, 18, 21, X, and Y have been developed for use in prenatal diagnosis. It is also possible to “paint” whole chromosomes using FISH. Using spectral karyotyping technology that combines mixtures of fluorochromes, it is now possible to produce a unique fluorescent pattern for each individual chromosome in 24 different colors. This technology permits the detection of complex chromosome rearrangements that cannot be seen with traditional cytogenetic techniques (Fig. 5-7).

Figure 5-7 Patterns of FISH probes in interphase cell preparations. The first figure shows a hypothetical locus-specific probe with a discrete signal. The middle figure shows a centromeric repeat probe with a larger, more diffuse signal. The figure on the right shows a whole chromosome painting probe with the diffuse pattern of overlapping domains making chromosome enumeration less reliable.

Prenatal Diagnosis

For an older woman, pregnancy may not be a time of joy but a time of anxiety. Advanced maternal age has a long-known association with an increasing risk of fetal chromosome abnormalities. Amniocentesis done at 16 weeks’ gestation followed by traditional karyotype analysis may take from 10 days to 2 weeks. The use of FISH for preliminary results can expedite a diagnosis and reduce the waiting time for results. Most geneticists and laboratories recommend that FISH not be used alone to make a management decision in pregnancy. FISH studies should always be confirmed with a final karyotype or at minimum correlated with abnormal ultrasound findings or an abnormal maternal serum analyte screen.

Contiguous Gene Syndromes

Contiguous gene syndromes are also known as microdeletion syndromes, or segmental aneusomy.¹⁰ These are deletions of a contiguous stretch of chromosome that usually involve multiple genes. The contiguous gene syndromes were first described in 1986 using classical cytogenetic methodologies. Using FISH, submicroscopic deletions can now be identified at the level of DNA; this has permitted the characterization of the smallest deleted region consistently associated with a syndrome, known as the critical region. By identifying the critical region of a syndrome, it is often possible to identify the specific genes that, when missing, are associated with the syndrome (Fig. 5-8). A recent compendium of deletion syndromes has reported 18 deletion and microdeletion syndromes spread over 14 chromosomes.¹³ Some of the more common deletion and mirodeletion syndromes and their clinical findings are shown in Table 5-2.

Figure 5-8 DiGeorge probe. This photo shows a peripheral blood sample from a patient tested for DiGeorge syndrome. The Tuple 1 probe binds to both copies of chromosome 22 as does the control. The child did not have the deletion causing DiGeorge syndrome.

Table 5-2 Deletion and Contiguous Gene Syndromes

Telomeres

Telomeres are structures that cap the tips of the long and short arms of chromosomes. They are composed of repeating sequences of TTAGGG and effectively prevent the ends of the chromosomes from fusing together. Telomere probes can be important in sorting out complex translocations that cannot be determined by traditional cytogenetic means. In addition, one of the findings of the Human Genome Project was that the chromosome regions next to telomeres are gene-rich. It has now been shown that submicroscopic subtelomeric deletions are responsible for a significant proportion of genetic morbidity.¹⁴

Single-Gene Disorders

In humans as with all diploid organisms, genes come in pairs on related or homologous chromosomes. The exception to this is the sex chromosomes, the X and the Y in the male. Single-gene disorders are described on the basis of the interaction of these pairs or alleles in the genotype and on how the interaction is expressed in the effect or the phenotype. When the alleles are not identical, the genotypes are heterozygous. When the alleles are identical, the genotype is described as homozygous. Gregor Mendel recognized that single-gene disorders are often recognizable and that these single-gene disorders segregate in families with predictable proportions; they have thus been referred to as mendelian traits.¹⁵ If a single-gene disorder manifests itself in the heterozygous state, the condition is inherited in a dominant manner. If the single-gene disorder is only inherited when both alleles are affected or altered, the condition is inherited in a recessive fashion. In addition, single-gene disorders are further characterized according to whether they are transmitted on the autosomes or the sex chromosomes.

Autosomal Dominant Inheritance

The following features characterize autosomal dominant inheritance:

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