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Features Departments Information |
 Joel M. Charrow, MD
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Missing genes JOEL M. CHARROW, MD aSpring 2000 THE PRESENCE OF MULTIPLE congenital anomalies in a child always raises the possibility that there is an underlying genetic disorder. In many instances, the pattern of anomalies is recognizable and is referred to as a syndrome. This article presents an overview of recent insights into the genetic basis of several well-known syndromes, which are now known to be related to submicroscopic deletions of chromosomes. Genetic disorders can result from a variety of mechanisms; perhaps most familiar to pediatricians are conditions like Down syndrome, which result from chromosomal duplication (the presence of an extra copy of a whole chromosome or part of a chromosome). Many single gene disorders (often referred to as mendelian disorders because of their autosomal dominant, recessive or X-linked inheritance) are also familiar, such as the Apert syndrome (autosomal dominant), Ellis van Creveld syndrome (autosomal recessive), and the Conradi syndrome (X-linked). Perhaps less often appreciated are those syndromes that result from deletion of a gene or cluster of genes that are contiguously arranged along a chromosome. WHEN MULTIPLE CONTIGUOUS GENES ARE MISSING Gene deletions have been observed in many of the single gene disorders. In some cases the phenotype associated with deletion of the gene is indistinguishable from that which results from a single base substitution; in other cases, the phenotype associated with deletion may be more severe (e.g., neurofibromatosis-1). It is the syndromes which result from deletions of multiple contiguous genes ("Contiguous Gene Syndromes") that are the subject of this review. Chromosome deletions have long been recognized as the cause of several clinically delineated syndromes. Well known among these are the Cri du Chat syndrome (Deletion 5p) and the Wolf-Hirschhorn syndrome (Deletion 4p). These were identified based on the discovery that part of one of the chromosomes was missing. More recently, smaller deletions have been observed in some children with well-recognized syndromes that were not previously associated with chromosome abnormalities, e.g., the Prader-Willi and Williams syndromes. The size of the deletions observed in these syndromes varies from individual to individual, and in many cases the deletion is not visible on routine chromosome analysis, even when the phenotype is clear and unambiguous. Clearly, the ability to detect such deletions depends on the resolving power of the chromosome analysis, and while resolution has improved significantly over the last three decades, deletions involving as many as several hundred genes may still go undetected by routine methods. GO FISH—HOW TO TELL WHEN SOMETHING TOO SMALL TO BE SEEN ISN’T THERE How then can we "see" a deletion which is smaller than the resolving power of our routine methods, i.e., a sub-microscopic deletion? The answer to this was found as new techniques for manipulating DNA were developed, and led to the procedure known as FISH (Fluorescent In Situ Hybridization). Using this technique we can actually "see" single genes located on specific chromosomes under the microscope. Conversely, we can also detect the absence of a single gene, using the same method.  FIGURE 1 Fluorescent in situ hybridization. A DNA probe, covalently bound to biotin, is hybridized to a denatured chromosome preparation. The probe binds to a homologous region on the chromosome. An avidin-bound fluor (FITC) is layered on top of the cells, and the avidin-FITC binds the biotin. The signal is amplified further by layering rabbit anti-avidin antibody (which binds the avidin-FITC), and then layering FITC-labeled anti-rabbit antibody on top. Fluorescence will be detected only where the DNA probe has hybridized to the chromosome. A FISH analysis begins in the same way as a regular chromosome analysis, and it is usually performed on circulating T-cells from a blood sample. However, instead of staining the chromosomes on slides, the chromosomal DNA is denatured, i.e., the double-stranded DNA is separated into two strands (without destroying the chromosome morphology). The next step depends on having a DNA probe to the specific chromosomal region of interest. For example, to study Williams syndrome we need a probe for the region7q11.2 (i.e., band 11.2 on the long or "q" arm of chromosome 7). A piece of DNA from this region is bound to a ligand, such as biotin. This biotin-bound probe is then allowed to hybridize (form a double-strand) with the denatured chromosomal DNA (Figure 1). If the "Williams syndrome region" is present on a chromosome, the probe will bind; if the region is absent, the probe won't bind. We can then try to "see" the probe by using a fluorescent dye which can bind to the biotin-labeled probe (in this example, the fluorescent dye is conjugated to the protein avidin, which specifically binds biotin). Further amplification of the signal can be achieved by further layering the chromosome with fluorescent antibodies. The end result is that a fluorescent signal can be seen under the microscope at the site where the DNA probe binds to the chromosome. Through the application of this technique, deletions of several chromosomal regions can be consistently demonstrated for the diagnosis of the associated syndromes. Descriptions and illustrations of some of these syndromes follow. WILLIAMS-BEUREN SYNDROME This well known syndrome is characterized by typical facial features (Figure 2), occasional hypercalcemia in infancy, supravalvular aortic stenosis, and moderate to severe mental retardation. The irides are often blue, and a particularly striking stellate pattern is sometimes present in them. The behavior of these children is often described as friendly and loquacious, reflecting the greater preservation of verbal IQ, vocabulary, social use of language and auditory memory, in contrast to their generally poor visual-motor integration and attention deficit disorder. Williams syndrome is quite common, with an estimated incidence of 1 in 10,000.  FIGURE 2 Facial features in unrelated three- and seven-year-old boys with Williams syndrome. Note the depressed nasal bridge, anteverted nares, long philtrum, and full lips with open mouth. Deletions involving 7q11.2 have been found in 95% of patients with Williams syndrome. Although the deletions are only rarely large enough to be seen with a regular chromosome analysis (Figure 3), they are easily detected with FISH (Figure 4).  FIGURE 3 Partial karyotype from a patient with Williams syndrome. There is a very small deletion at 7q11 (arrow). Deletions of this size and smaller may not be detected on regular chromosome analyses. These deletions typically include the elastin gene (ELN) and the nearby LIM kinase-1 and RFC2 genes. Other genes may be involved as well. The deletions are almost always sporadic; only rare instances of dominant inheritance have been reported.  FIGURE 4A Fluorescent in situ hybridization (FISH) of normal chromosomes using a probe for elastin (which is in the Williams syndrome region and fluoresces pink) and a "control" probe for a more distal region on the long arm of chromosome 7 (green dots). The control probe helps identify the number 7 chromosomes; because the elastin probe has hybridized to both #7s, there is no deletion in this individual. Williams syndrome is truly a contiguous gene syndrome. Deletions of the elastin gene alone and mutations within ELN result in isolated supravalvular aortic stenosis without the other features of Williams syndrome. PRADER-WILLI SYNDROME The Prader-Willi syndrome is probably best known because of the morbid obesity and voracious appetite that is associated with it. Infants with PWS are often hypotonic early in infancy and may fail to thrive during the first six months of life because of poor feeding. Appetite later improves, and binge eating and extreme food seeking behaviors are common. These children may be mildly mentally retarded; less commonly, moderate or severe retardation is present. The hands and feet are small, as are the genitals, and hypogonadism is common. Distinctive facial features make recognition of the syndrome relatively easy in most cases (Figure 5).  FIGURE 5 A 3-year-old markedly obese boy with Prader-Willi syndrome. Chromosome deletions of 15q11–13 may be observed in as many as 50% of patients with PWS; with the addition of FISH, deletions are found in close to 70%. Interestingly, the deletion is always on the chromosome 15 inherited from the father. This tells us that the PWS region is imprinted, i.e., expression of the paternal allele is necessary to prevent the syndrome. Most of the patients who do not have deletions have uniparental disomy, i.e., both of their number 15 chromosomes are inherited from their mothers, so, again, there is no paternal copy. Finally, mutations in genes that control imprinting of the genes in the PWS region are a rare cause of this syndrome. FISH studies of 15q11-13 can be used to detect deletions in PWS, and help confirm the diagnosis. However, because uniparental disomy and imprinting mutations can also lead to impaired expression of the paternal allele, a normal FISH study (i.e., no deletion) does not rule out the diagnosis. ANGELMAN SYNDROME Facial features are also characteristic in this syndrome, with microcephaly, prominence of the mandible, maxillary hypoplasia, deep-set eyes and a large mouth. It is associated with severe mental retardation, jerky and ataxic limb movements, paroxysms of inappropriate laughter, and the development of seizures by 18 to 24 months of age. As in PWS, deletion of 15q11–13 is found in approximately 70% of patients with Angelman syndrome. However, in this condition, the deletion is found on the maternally inherited chromosome. A single gene, UBE3A (the E6-associated protein ubiquitin protein-ligase gene), is responsible for the phenotype, and deletion of this gene alone, or mutations within this gene, can produce the syndrome. As in PWS, the syndrome can also result from uniparental disomy (here both copies of chromosome 15 are from the father) and from mutations in the genes that control imprinting. DIGEORGE SEQUENCE—VELOCARDIOFACIAL SYNDROME (SHPRINTZEN) — CATCH 22 This contiguous gene syndrome demonstrates the enormous phenotypic variability that can occur with deletions. It is also extremely common, with an estimated incidence of 1 in 4,000. At the one end of the spectrum is the complete DiGeorge syndrome (Table 1), with its associated congenital heart disease, hypoparathyroidism, and T-cell immune deficits. These result from abnormal development of the third and fourth pharyngeal pouches and adjacent neural crest tissue, which affect the development of the outflow tract of the heart, the parathyroids, and the thymus. The cardiac anomalies are generally conotruncal defects, including right aortic arch, interrupted aorta, truncus arteriosus, tetralogy of Fallot, and even isolated ventricular septal defect.
At the other end of the spectrum of this disorder is the velocardiofacial syndrome (Table 2), comprised of distinctive facial features (long nose with bulbous tip) and varying degrees of palate malformations, including submucous cleft, overt cleft palate, and palatal incompetence without cleft.
Deletions of 22q11 are found in 90% of patients with DiGeorge syndrome. Similar deletions are found in children with velocardiofacial syndrome, isolated tetralogy of Fallot, or isolated velopharyngeal incompetence. The same deletion is observed in as many as 10–25% of parents of children with the syndrome; these adults are most often asymptomatic, but again they demonstrate the phenotypic variability and dominant inheritance of this disorder. Although the size of the deletion varies, it does not seem to correlate with the severity of the phenotype, nor do the presence or absence of specific loci within the region correlate with specific aspects of the syndrome. Deletions involving 10p13 account for at least some of the cases lacking the 22q11 deletion. MILLER-DIEKER SYNDROME Until recently this syndrome was thought to be an autosomal recessive disorder because of the occurrence of multiple affected siblings in several families. It is now clear that the syndrome is a contiguous gene syndrome resulting from deletion of 17p13.3. The deleted region includes the lissencephaly gene, LIS1, which is responsible for the type 1 lissencephaly (smooth brain) observed in these children. The brain may entirely lack gyri (Figure 6), or may have shallow and thick gyri (pachygyri). This brain abnormality may be seen in isolation in children with LIS1 mutations or deletions.  FIGURE 6 Mid-sagittal and axial brain MRIs of a four-month-old child with type I lissencephaly and Miller-Dieker syndrome. Note the near complete absence of gyri and sulci. The deletions in children with the Miller-Dieker syndrome include genes adjacent to LIS1, accounting for the very characteristic facial features seen in this devastating syndrome: microcephaly, bitemporal indentations, widely spaced eyes, and furrowing of the forehead, especially when crying (Figure 7). Life expectancy is usually less than two years.  FIGURE 7 Facial features in the Miller-Dieker syndrome, demonstrating bitemporal narrowing, tall forehead, small nose with depressed nasal bridge and anteverted nares, and thin vermilion border of upper lip. The recurrences among siblings and the apparent autosomal recessive inheritance of this syndrome in some families can be accounted for by balanced translocations in one of the parents, with transmission of an unbalanced chromosomal complement to the affected siblings (each of whom had a deletion of the critical region on 17p resulting from the translocation). TRICHORHINOPHALANGEAL SYNDROMES Disruption of a putative zinc-finger transcription factor (TRPS1) is associated with characteristic facial features including sparse scalp hair, tall forehead, pear-shaped nose with long philtrum, and short stature (Figure 8). Intelligence is normal, and the metacarpals and metatarsals are short, with a distinctive radiographic appearance (cone-shaped and ivory epiphyses). This syndrome, the trichorhinophalangeal syndrome type I (TRP I), is inherited as an autosomal dominant.  FIGURE 8 Trichorhinophalangeal syndrome type I. [Above] Facial features in a three-year-old boy with a long, pear-shaped nose, long philtrum and prominent ears. Scalp hair is relatively sparse. [Below] Brachydactyly in an affected adult woman and her three-year-old child; note widening of the proximal interphalangeal joints.  In the Langer-Giedion syndrome (trichorhinophalangeal syndrome, type II), there is deletion of the TRPS1 gene and adjacent contiguous genes located at 8q24.12. As a result of the loss of the contiguous genes, the phenotype includes microcephaly, mental retardation, redundant skin, and multiple cartilaginous exostoses, in addition to the features of TRP I described above. The exostoses can be accounted for by the inclusion of EXT1 in the deletion. This gene is one of several which, when deleted or mutated, causes hereditary multiple exostoses. CONCLUSION The addition of FISH to the diagnostic armamentarium has made the routine laboratory diagnosis of contiguous gene deletions possible and has greatly enhanced our ability to provide anticipatory guidance, genetic counseling, and prenatal diagnosis for these conditions. FISH has also been applied to the identification of single gene deletions, identification of chromosome rearrangements (Figure 9) and marker chromosomes, and a number of applications in cancer cytogenetics. Finally, FISH has also been applied to interphase cells, where the chromatin is not condensed, and has made possible the rapid detection of common trisomies and sex-chromosome complement. The DNA probes required for FISH are commercially available for many of these applications.  FIGURE 9 Use of FISH to determine the chromosomal origin of a duplication arising from an unbalanced translocation. Utilizing FISH and other molecular genetic techniques has led to the recognition of many other contiguous gene syndromes not covered here (e.g., Smith-Magenis syndrome, steroid sulfatase deletion syndrome, Rubinstein-Taybi syndrome and Kallman syndrome). It will not be surprising if many other clinically defined syndromes of unknown etiology are also found to be the result of sub-microscopic deletions as well. FOR FURTHER READING 1. Jones KL: Smith’s Recognizable Patterns of Human Malformation, 5th Edition, W.B. Saunders, Philadelphia, 1997. 2. McKusick VA: Online Mendelian Inheritance in Man, the National Center for Biotechnology Information, http://www3.ncbi.nlm.nih.gov/Omim/. 3. Shapira SK: An update on chromosome deletion and microdeletion syndromes. Current Opinion in Pediatrics, 1998;10:622–627. 4. Ferguson-Smith MA, Andrews T: Cytogenetic analysis, in Principles and Practice of Medical Genetics, 3rd ed, Rimoin DL, Connor JM, Pyeritz RE (eds.), New York: Churchill-Livingstone, 1996. |