Genetics, Genetic Counseling, and Prevention

Identifying Visible Chromosome Abnormalities: The Karyotype

Fig. 2.4 shows the natural appearance of the stained chromosomes at early, middle, and later stages of mitosis. It would obviously be difficult to count these chromosomes or to distinguish their individual structure from such preparations. To obtain adequate preparations for the study of chromosome number and morphology, the cultured cells are treated with an agent that blocks the spindle formation and thus leads to the accumulation of cells at the metaphase of mitosis. These cells are then exposed to a hypotonic solution that spreads the unattached chromosomes, allowing for preparations such as those shown in Fig. 2.5 . Various techniques, such as trypsin treatment and Giemsa staining, can be used to allow for the identification of individual chromosomes. The development of synchronized culture techniques that allow evaluation of chromosomes in prophase and prometaphase have greatly enhanced the ability to detect subtle abnormalities and have expanded our understanding of the impact of chromosomal rearrangement on morphogenesis. A chromosome analysis using this technique is a high-resolution analysis ( Fig. 2.6 ). Banding techniques applied on metaphase or prometaphase preparations allow the recognition of each of the individual chromosomes, aneuploidies, and loss or gain of chromosome fragments larger than 5 Mb in standard resolution and 3 Mb in high-resolution karyotypes. All chromosome studies require cell culture in advance.

Identifying Smaller Genomic Imbalance: FISH, CGH, Arrays, and MLPA

Two technologies that allow detection of more subtle changes in copy number in the genome are fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). In FISH, fluorescent-labeled probes of known DNA sequence are hybridized to chromosomes that are fixed on a slide and denatured in place ( in situ ), allowing the probe to attach to its complementary sequence. When viewed with a wavelength of light that excites the fluorescent dye, a colored signal is generated, allowing localization of the probe. FISH probes may consist of contiguous genomic sequences, parts of chromosomes, or whole chromosomes. Depending on the probe and the clinical question, FISH may be performed on interphase instead of metaphase cells; this would offer advantages in some clinical situations such as prenatal diagnosis. Interphase FISH obviates the need for cell culture and may be performed rapidly; however, it will not locate a targeted DNA sequence in a specific chromosomal region. CGH is based on FISH technology. DNA from one sample is labeled with a red fluorescent dye, whereas DNA from another is labeled in green. The two are mixed in equal amounts and “painted” on normal human chromosome preparations. The ratio of red-to-green fluorescence along each chromosome is measured. Deviations from the expected 1:1 ratio of red to green will be detected as a change in the color signal in that region documenting gain or loss of copy number. Chromosomal CGH has been used to identify the chromosomal origin of small chromosome fragments of unknown origin (markers). It has also been used to identify visible extra bands in a karyotype that cannot be identified based on the banding pattern itself. Even greater resolution can be achieved using known DNA sequences instead of whole chromosomes as hybridization targets. DNA sequences can be printed on a chip such that small fragments of DNA may be interrogated. If the printed sequences overlap, in a so-called tiling-path array, coverage of the entire genome may be achieved. A variety of different probes are in clinical usage, including large ones derived from bacterial artificial chromosomes (BAC array), small ones consisting of oligonucleotide sequences (oligo-array), and very small single nucleotide polymorphisms (SNPs) containing sequences (SNP array). Array technology does not require cell culture. Small amounts of DNA can suffice, as DNA can undergo preamplification ( Fig. 2.7 ).

Multiple ligation probe amplification (MLPA), a variation of the multiplex polymerase chain reaction (PCR), permits up to 40 targets to be amplified with only a single primer pair. MLPA is often used to interrogate dosage of multiple fragments in a specific genomic region known to have variable size deletions, or in all the subtelomeres in one reaction. Because it costs much less than chromosomal microarrays, MPLA is used to confirm array results and to verify the presence or absence of an abnormality in a parent.

Abnormal Number of Chromosomes (Aneuploidy)

Although much is being learned about the etiology of faulty chromosomal distribution, one clear recognized factor is older maternal age. This applies especially to the autosomal trisomy syndromes and to the sex chromosome aneuploidy, XXX and XXY. Fig. 2.10 shows the progressive increase in the frequency of live-born infants with Down syndrome during the later period of a woman’s reproductive life. The frequency of aneuploidy detected by amniocentesis at 14 to 16 weeks’ gestation is appreciably higher because some of the aneuploid conceptuses detected at this early stage in gestation would normally abort spontaneously or die in utero later in pregnancy.

The timing of the error in chromosome distribution can seldom be stated with assurance from a routine karyotype, although molecular techniques, as is discussed subsequently, have permitted detailed investigation of this issue in certain aneuploidy states. Numerical errors may result from altered chromosomal segregation in the cells that will give rise to the germ cells (gonadal mosaicism), or in either the first or second division of meiosis leading to an abnormal chromosome number in the egg or sperm (nondisjunction), or during the first divisions of the newly formed zygote. Errors in the assortment of chromosomes may also occur later in embryogenesis, giving rise to somatic mosaic individuals who have two populations of cells from the standpoint of chromosome number. Mosaicism also develops when a trisomic conceptus “self-corrects” and loses one copy of the trisomic chromosome in early cell division, thus establishing a normal cell line along with the aneuploid cell line. This process has been termed trisomic rescue . Individuals who are mosaic for a condition show every gradation of the phenotype associated with that chromosomal abnormality, from a pattern indistinguishable from complete aneuploidy to near-normal appearance and function. In general, the degree of mosaicism present in the peripheral blood is not, in and of itself, that helpful in predicting prognosis. Detection of mosaicism may require the sampling of more than one tissue. Identification of the parent of origin of individual chromosomes has shed some light on the source of the extra or deleted chromosome and the stage of cell division during which accidents leading to aneuploidy occur. In conceptuses and live-born individuals with 45,X, the chromosome that is deleted is usually paternal in origin. This is consistent with the observation that maternal age is not related to a 45,X karyotype in the fetus. By contrast, the extra chromosome in trisomy 21 is of maternal origin in 95% of cases. Most of the maternal errors involve nondisjunction in meiosis I. Of the paternally derived chromosomes, most represent errors in meiosis II. Similarly, the extra X chromosome in 47,XXX females is usually maternally derived. In 47,XXY the source of the extra chromosome appears to be equally divided. In those cases of 47,XXY and 47,XXX with a maternally derived extra chromosome, increasing maternal age correlates with errors in the first meiotic cell division, but not with errors in meiosis II or in postzygotic events. The precise etiology of nondisjunction is unknown; however, evidence is accumulating that mammalian trisomies may be a consequence of abnormal levels or positioning of meiotic crossovers (recombination events). Between 1% and 5% of sperm from chromosomally normal men are aneuploid. Indirect estimates from spontaneous abortions and studies of embryos from in vitro fertilization clinics have suggested an aneuploidy rate of nearly 25% in oocytes.

Structural Chromosomal and Genomic Rearrangements

In addition to resulting from errors in chromosome number, genetic imbalance can result from chromosomal rearrangement (see Fig. 2.8 ). A break in one chromosome may result in loss or gain of information (deletion or duplication). If more than one chromosome breaks, rearrangement of the resulting pieces may take place, creating a translocation. Reciprocal translocations always involve two chromosomal fragments, including two different telomeres, and can be recognized with techniques that will identify the unique subtelomeric sequences of each chromosome, such as subtelomeric FISH or MLPA. Robertsonian translocations occur among acrocentric chromosomes, in which the small arms that contain redundant genetic sequences are lost in the rearrangement, leading to a derivative chromosome containing the long arms of two different chromosomes in a karyotype with 45 chromosomes. The genome is still functionally balanced. An individual can have a translocation between chromosomes with no evident problem as long as he or she has a balanced set of genes. Only in cases in which the breakpoints cause a cryptic deletion or involve an important exonic or regulatory sequence will the “balanced” rearrangement be associated with an altered phenotype. However, as illustrated in Fig. 2.11 , a balanced carrier of a translocation has a significant risk of producing unbalanced germ cells during the meiotic reduction division, meiosis I. Should a germ cell receive the translocation chromosome as well as the normal 21 chromosome from the same parent, the resulting zygote would be trisomic for most of chromosome 21. Such individuals generally have Down syndrome. About 4% of patients with Down syndrome have 46 chromosomes, with the extra set being attached to another chromosome. Similarly, a small proportion of patients with the trisomy 18 syndrome or the trisomy 13 syndrome have the extra set of genes attached as part of a translocation chromosome.

Some patterns of malformation result from a deletion (or duplication) of chromosomal material in which the missing (or extra) piece is so small that routine chromosome analysis cannot detect the abnormality. Such conditions are referred to as microdeletion (microduplication) syndromes to denote the fact that the phenotype is a consequence of imbalance in dosage of several genes that lie next to each other along a chromosome. If several genes in the deleted (or duplicated) segment are responsible for the phenotype, the condition may be designated a contiguous gene disorder. However, the phenotypes of some microdeletions (microduplications) are actually the consequence of imbalance in a single gene in the rearranged interval. These deletions/duplications may be considered monogenic disorders. FISH, MLPA, and array technologies are techniques used to identify these submicroscopic rearrangements. Breaks in chromosomes can arise through various mechanisms. However, there are recurrent rearrangements that occur because the regional structure of the genome contains low copy repeats (LCRs), also called segmental duplications, that predispose to nonallelic homologous recombination (NAHR), leading to deletion or duplication of the single copy sequences flanked by these LCRs ( Fig. 2.12 ). NAHR produces some of the most common microdeletion and microduplication syndromes. Many are associated with recognizable patterns, not only because they are frequent but also because the deleted (or duplicated) segments are identical.

Another type of chromosomal abnormality that can lead to genetic imbalance is maldivision at the centromere during mitosis, leading to the formation of an isochromosome (see Fig. 2.8 ). The cell receiving the isochromosome has an extra dose of either the long or short arm of the parent chromosome and is missing the set of genes on the opposite arm. Isochromosome Xq accounts for roughly 10% of the cases of Turner syndrome in live-born female infants.

Interpretation of the Causality of Genomic Imbalance

Whereas the loss or gain of visible chromosome fragments containing hundreds of genes is almost always the cause of an abnormal phenotype, smaller imbalances are most often part of human normal variation. The frequency of copy number variants (CNVs) in the genome is very high and, in most cases, reflects normal dosage variation not associated with phenotype. Knowledge of the consequences of dosage imbalance throughout the genome is currently incomplete. This often makes the interpretation of array findings very difficult. In addition to the medical literature, web-based tools such as DECIPHER may be of assistance in determining if CNV is benign or pathologic.

Autosomal Trisomy Syndromes

Chromosomal studies are warranted in all individuals suspected of having an autosomal trisomy syndrome to determine whether full trisomy (47 chromosomes) or an unbalanced robertsonian translocation is involved. If a full trisomy is identified, the risk for recurrence is roughly 1%. For women 35 years of age and older, the risk is based on the maternal age at delivery in the subsequent pregnancy. For trisomy 21, parental karyotypes are suggested only if a second child in the same sibship has an identical trisomy. In this rare circumstance mosaicism in one of the parents may be detected in as much as 38% of families if a diligent search is made. The presence of a second- or third-degree relative with a similar trisomy can be accounted for by chance alone and does not appear to increase the risk for recurrence.

Should an unbalanced robertsonian translocation be identified, both parents must be evaluated to determine if either one is a balanced translocation carrier, a finding in approximately one-third of cases. The recurrence risk for parents with normal chromosomes is very small (probably less than 1%) and reflects the unlikely possibility of gonadal mosaicism that cannot be identified by peripheral blood karyotype. The recurrence risk for a carrier parent is obviously increased, but it is often different, depending on the gender of the carrier parent, and less of a risk than the theoretic possibilities might indicate because of the frequent prenatal lethality of autosomal trisomies (see Fig. 2.11 ).

Other Chromosomal Disorders