The basic process of morphogenesis is genetically controlled. However, the ability of an individual to reach his or her genetic potential with respect to structure, growth, or cognitive development is affected by environmental factors in both prenatal and postnatal life. Review of the etiologies of those structural abnormalities and syndromes for which an etiology is known indicates that the majority of malformations and syndromes appear to be genetically determined. The purpose of this chapter is to outline the most prevalent mechanisms through which genetic abnormalities impact morphogenesis, to discuss the techniques that are currently available for genetic testing, to suggest genetic counseling for each of these abnormalities, and to discuss approaches to prevention.
The haploid human genome contains just over 20,000 protein-coding genes, which are far fewer than had been expected before sequencing. Only about 1.5% of the genome codes for proteins. The rest consists of noncoding RNA genes, regulatory sequences, introns, and noncoding DNA.
Genes come in pairs. The great majority of these genes are distributed in the 46 chromosomes that are found in the nucleus of the cell. A few genes reside in the cytoplasm inside the mitochondria, the energy-producing apparatus of the cell. Genetic abnormalities may be grossly divided into those that affect gene dosage (chromosomal and genomic abnormalities), those that involve changes (mutations) in the actual genes themselves (single-gene disorders), and those that create a susceptibility to developmental errors that is then modified by other genes and factors in the environment (polygenic and multifactorial inheritance). The frequency with which each of these genetic mechanisms contributes to malformation and disease depends on the time in development at which inquiry is made. For example, roughly half of all first-trimester miscarriages are a consequence of chromosomal abnormalities, whereas only 6 of 1000 live-born infants are similarly affected. Fig. 2.1 provides a perspective as to the frequency with which each mechanism contributes to birth defects or human disease over the lifetime of a population. Each of these problems is considered separately as it relates to malformation, especially multiple defect syndromes. Recommended genetic counseling is presented at the end of each section.
Genetic Imbalance Caused by Gross Chromosomal Abnormalities and Submicroscopic Genomic Imbalance
The 46 normal chromosomes consist of 22 homologous pairs of autosomes plus an XX pair of sex chromosomes in the female or an XY pair in the male. Normal development depends not only on the gene content of these chromosomes but on the gene balance as well. An altered number of chromosomes most commonly arises because of a fault in chromosome distribution at cell division. During the gametic meiotic reduction division ( Fig. 2.2 ), one of each pair of autosomes and one of the sex chromosomes are distributed randomly to each daughter cell, whereas during mitosis ( Fig. 2.3 ), each replicated chromosome is separated longitudinally at the centromere so that each daughter cell receives an identical complement of genetic material. Abnormal segregation in meiosis or mitosis will lead to an incorrect number of chromosomes (aneuploidy) in daughter cells. In addition, a piece of a chromosome can be deleted, duplicated, inverted, or exchanged between two chromosomes.
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.
The Impact of Chromosomal and Genomic Imbalance during Development
Fig. 2.8 illustrates some of the mechanisms that can lead to genetic imbalance (too many or too few copies of normal genes) as a consequence of chromosomal rearrangement and maldistribution. Such cytogenetically visible abnormalities occur in at least 4% of recognized pregnancies. Most of these imbalances have such an adverse effect on morphogenesis that the conceptus does not survive. Smaller imbalances more likely result in surviving individuals with variable dysmorphology and developmental disability. Fig. 2.9 summarizes the frequency and types of visible chromosomal abnormalities found in newborns and spontaneous abortuses. Approximately 50% of these have a chromosome abnormality compared with 0.5% of live-born babies. The nature of the abnormalities detected in live-born infants differs from those seen in abortuses, with sex chromosomal aneuploidy and trisomy 21 (Down syndrome) accounting for most of the anomalies observed in live-born infants because these are least likely to have an early lethal effect. It has been estimated that only approximately 1 in 500 45,X conceptuses survives to term compared with 4% of trisomies 18 and 13, and 20% of trisomy 21 conceptuses. Some data suggest that survival is impacted by the presence of a normal, as well as an aneuploid, cell line (mosaicism). The Human Genome Project has identified that the genome is in clumps with some chromosomes (such as 19 with 1621 known genes) being gene-rich and others (such as the Y with 251 genes) gene-poor. Autosomes 21, 18, and 13 are relatively gene-poor, perhaps contributing to their in utero survival. In general, smaller genomic imbalances will more often be viable, presenting with major and/or minor malformations, intellectual disability, and/or abnormal behavior. Microdeletions and microduplications as a group are found more often than aneuploidy in children presenting postnatally with delayed psychomotor development.
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.
Incidence of Chromosomal Abnormalities and Genomic Rearrangements in Patients with Intellectual Disability
Surveys of the incidence of visible chromosomal abnormalities in newborns have documented that roughly 1 in 520 normal individuals has a balanced structural chromosomal rearrangement, whereas 1 in 1700 newborns has an unbalanced rearrangement. The incidence of microdeletions/duplications in the general population is unknown. Systematic surveys of undiagnosed cases of intellectual disability and multiple structural defects have documented an 8% incidence of visible chromosome abnormalities. High-resolution chromosome analysis identifies an additional 1.1% of patients evaluated for similar indications. A 7.4% detection rate for submicroscopic chromosome rearrangements among individuals with moderate to severe intellectual disability of unknown etiology and a 0.5% rate for mild intellectual disability have been documented based on the use of FISH or MLPA probes, which recognize the unique subtelomeric sequences of each chromosome. More recently array technologies have increased the detection rate of pathogenic genomic rearrangements from 14% to 20%, depending on whether these numbers are assessed in patients with previous genetic testing or no previous genetic testing.
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.
Indications and Sequence of Chromosomal and Genomic Studies
The utility of array technology as a “first round” test has been firmly established, with recognition of a much higher detection rate of pathogenic genomic imbalance than that revealed by even high-resolution chromosome analysis. Because of its lower cost, standard karyotype remains the preferred test in specific situations such as clinical suspicion of specific aneuploidies or syndromes associated with large genomic rearrangements. In addition, a karyotype is needed for the identification of balanced rearrangements (translocations and inversions), which are not detected by any technology addressing dosage. FISH with unique sequence probes is still the least costly way to confirm the diagnosis in well-known phenotypes such as Williams syndrome (del 7q11.2) shown in Fig. 2.13 or the velocardiofacial syndrome (del 22q11.2). Multitelomere FISH remains the optimal way to diagnose small reciprocal translocations. With respect to mosaicism, arrays are not able to detect levels below roughly 20%. Karyotyping or FISH may be required, including examining several different tissues and scoring large numbers of cells.
Genetic Counseling for Chromosomal and Genomic Abnormalities
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
Chromosome analysis is still the optimal way to diagnose Turner syndrome. Although a wide variety of chromosomal rearrangements are known to produce the phenotype (including X/XX and X/XY mosaicism, X, iso X, or X, deleted X), the recurrence risk for these arrangements is low to negligible. The finding of a Y-bearing cell line suggests an increased risk for malignant tumor in the dysgenetic gonad, which should be removed.
Any Case with a Visible Deletion, Duplication, or Unbalanced Translocation
In this situation, chromosome studies should be done on both parents to rule out a rearrangement such as a pericentric inversion, a balanced translocation, or, in rare cases, an insertion, that could predispose to recurrence of the abnormality. If parental karyotypes are normal, as is the case in the majority of families, the recurrence risk is low. If a parental rearrangement is identified, the theoretic risk for recurrence is increased. Other family members may be at risk. For some of the more common rearrangements, empiric risk figures are available in the literature. The actual risks often do not coincide with the theoretic risk as has been previously reviewed (see Fig. 2.11 ). In the case of balanced translocation carriers, the previous reproductive experience of the couple must be considered in counseling.
Microdeletion and Microduplication Syndromes
Although microdeletion syndromes are chromosomal abnormalities, because the problem that produces the phenotype is genetic imbalance rather than genetic mutation and because the abnormality is identified using molecular cytogenetic methodology, from a counseling standpoint the conditions behave like dominantly inherited Mendelian disorders. The majority of cases represent de novo events that carry a negligible risk for recurrence for unaffected parents and a 50% risk for the affected individual’s offspring. Evaluation of parents using FISH analysis, MLPA, or focused high-resolution cytogenetics is recommended, because vertical transmission of microdeletion syndromes is reported. Many, but not all, parents with microdeletions also express the phenotype to some degree. Recurrence risk for the genomic anomaly in these individuals is 50% for each subsequent pregnancy, but the severity of the phenotype that might ensue is often unpredictable, thus posing a challenge for genetic counseling.
Identification of a new microdeletion or microduplication—neither previously found in the normal population with significant frequency nor previously reported in affected patients with concordant phenotypes—is a challenge for interpretation. In case the CNV contains genes, or has a significant size, parents should be tested. If the CNV is inherited from a normal parent and no studies point to pathogenicity, it is usually considered a benign variant. If de novo , the assumption of causality can only be based on gene content, animal studies, and the concordance of the specific gene loss or duplication with a recognized phenotype. Unfortunately, many microdeletions or microduplications will remain as CNVs of uncertain significance, and future experience will confirm their association with phenotype or their benign nature. The literature and public databases should be searched for previously reported cases before providing the family with significant information on prognosis and natural history.
Genetic Imbalance Caused by Single-Gene Disorders
Genes located on the X chromosome are referred to as X-linked genes and those on the autosomes as autosomal genes. A human being is a diploid organism with two sets of chromosomes, one set from each parent. Each pair of chromosomes will have comparable gene determinants located at the same position on each chromosome pair. The pair of genes may be referred to as alleles, or partners, which normally work together. Thus, with the exception of the genes of the X and Y chromosomes in the male and those of the mitochondria, each genetic determinant is present in two doses, one from each parent. Biallelic expression of most genes is the common rule. However, for most genes on the X and for close to a hundred genes on the autosomes, only a single copy of the gene is actively expressed (monoallelic expression). A mutant gene indicates a changed gene. A major mutant gene is herein defined as a genetic determinant that has changed in such a way that it can give rise to an abnormal characteristic. If a mutant gene in a single dose produces an abnormal characteristic despite the presence of a normal allele (partner), it is referred to as “dominant” because it causes abnormality even when counterbalanced by a normal gene partner. A mutant gene that causes an abnormal characteristic when present in double dosage (or single dosage without a normal partner, as for an X-linked mutant gene in the male) is referred to as “recessive.” These principles, set forth diagrammatically in Fig. 2.14 , reflect Mendelian laws of inheritance, which equate the presence of an altered gene or pair of genes with a phenotype or trait. As more is learned about the molecular biology of mutant genes, the distinction between dominant and recessive genes has blurred. Dominant mutations impact development through a variety of mechanisms. Loss of one copy of the gene (haploinsufficiency) may reduce by half the gene product resulting in functional alteration of development. Mutations may also create proteins with either an increased function (gain of function mutations) or a totally new function (dominant negative mutations) that will interfere with normal development as well. Interestingly, mutations causing haploinsufficiency in the gene may cause one phenotype and those resulting in gain of function, a completely different one. The various forms of osteogenesis imperfecta are good examples of types of dominant mutations. Because collagen is a triple helical molecule, mutations that give rise to one abnormal procollagen molecule will impact the final assembly process and produce a severe skeletal phenotype, whereas mutations that reduce but do not alter the gene product typically result in mild fracturing. Recessive mutations also often serve to reduce the quantity of product made by half; however, many biologic systems are forgiving of quantitative decrease in gene function—hence the silence of recessive mutations when present in single copy (heterozygosity). Hurler syndrome is an example. Half of the normal amount of activity of alpha iduronidase has no effect on the individual with the altered gene; however, the enzyme deficiency resulting from a double dose of the altered gene produces a severe phenotype.