© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
On the trail of genetic culprits in Williams syndrome
Howard Hughes Medical Institute, University of Utah, Building 533, Salt Lake City, Utah 84112, USA
Received 1 July 1996; accepted 13 December 1996
KEYWORDS Genetics; Williams syndrome
In times past—in point of fact, not very long ago: only in the years after the genetic code was deciphered—the genetic basis of certain human diseases could only be extrapolated from knowledge of biochemical correlates. A critical enzyme was missing, for example, as in phenylketonuria, or a defective hemoglobin resulted in sickle-cell anemia. Determining the DNA sequence of a defective gene was a worthwhile exercise though, because if the basis for a disorder could be identified at the coding level, investigators might be able to devise novel approaches to prevention or treatment. But for hundreds, even thousands, of heritable disorders, not to speak of common and apparently sporadic illnesses, first causes remained mysterious; with no known protein culprit, then, how could the responsible (gene)s be discovered and the physiological consequences of mutations be evaluated?
One solution—‘molecular genetics’, a major advance in molecular biology, using recombinant DNA technology to map the human genome with anonymous (non-gene) DNA markers—has gained astonishing speed within the past 15 years [1]. Localization of markers—now available in thousands—to chromosomes permits investigators to track inherited diseases in pedigrees according to linkage (i.e., co-inheritance) of a defective allele to a marker of known location [2]. As regards cardiovascular diseases, the linkage approach led to the mapping of three loci that can carry mutations responsible for long QT syndrome, on chromosomes 11p15.5, 7q35–36, and 3p21–24, respectively [3–5]. Once the site on a chromosome is determined, techniques of manipulating fragments of DNA come into play until a specific gene in the region can be shown to contain abnormalities in structure or sequence in affected family members as compared to normal individuals. Using this strategy, known as ‘positional cloning’, we eventually identified the LQT genes on 7q as HERG, a putative cardiac potassium channel gene, and on chromosome 3p as SCN5A, a cardiac sodium channel gene [6, 7].
In the course of this recent explosion of genetic information, medical scientists have come to understand that common diseases not generally thought of as ‘inherited’ do, in fact, answer ultimately to multiple genetic factors that may be influenced by environment: cancers, certainly; many cardiac diseases probably; and even infectious diseases possibly, in terms of susceptibility. The more we learn about genes involved in inherited illnesses, however, the more light can be shed on related, seemingly sporadic disorders that affect the same organs or systems. Here, I describe our efforts to identify the genetic basis of Williams syndrome (WS), an inherited condition involving cardiovascular anomalies of profound significance not only to the patients who suffer them and to their families, but also for what the genes associated with them can tell us about vascular physiology in general.
A complex developmental disorder that affects approximately one in 20 000 live births, WS exhibits a remarkable phenotype that is readily distinguished from other disorders involving mental retardation. Features of WS include congenital heart and vascular disease, dysmorphic facial features, infantile hypercalcemia, mental retardation and specific neurobehavioral traits (gregarious personality, good verbal skills, poor visuomotor integration). As an example, many WS patients have difficulty following simple directions. Most individuals with WS have mild to moderate mental retardation (mean IQ 55–60), but some have borderline normal intelligence or severe mental retardation [8]. The characteristic personality includes excessive friendliness, loquaciousness, sensitivity to the feelings of others, and extreme anxiety to please. As a result, WS children make friends easily, but their need for socialization often becomes troublesome during adulthood (a list of features associated with WS is shown in Table 1). The phenotypic description was not recognized as a discrete clinical syndrome until the 1960's; at that time its molecular cause was thought to be related to calcium or vitamin D metabolism because clinicians had been noting a prevalence of hypercalcemia, associated with vitamin D supplementation, among children with the features described in the table [9].
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A common feature of WS is supravalvular aortic stenosis (SVAS). This vascular disorder, which can also be inherited as an isolated, autosomal dominant trait, was recognized clinically in the 19th century. SVAS causes narrowing of large, elastic vessels like the aorta and pulmonary arteries [10, 11]; it often presents in childhood, and if not corrected by surgery, can lead to heart failure and death. The histopathology of SVAS includes disease of the intima and media of the affected arteries, including disruption of elastic fibers, hypertrophy of smooth muscle cells, disruption of the intima, and intimal proliferation of smooth muscle in fibroblasts.
Until recently relatively little was known about the pathogenesis of SVAS. We used genetic linkage analysis of two kindreds segregating an allele for autosomal dominant SVAS to map the disease locus to chromosome 7q11.23, a region known to incorporate the gene encoding elastin [12]. The human elastin gene encodes a protein of about 786 amino acids and the polypeptide chains are cross-linked to give rise to elastin fibers, which are responsible for its elastic properties. To test the hypothesis that mutations in elastin cause this disorder, we screened DNA from SVAS patients for mutations in the elastin gene. In one family, we discovered that a balanced translocation disrupted the elastin gene, leading to a new stop codon on exon 28 (the human elastin gene has 34 exons) [13]. A deletion beginning at elastin exon 28 and extending throughout the remaining exons of the gene was identified in a second family [14]. Olson and colleagues, after independently mapping SVAS to chromosome 7q11, subsequently showed that an intragenic deletion of elastin was associated with the disease in another family [15]. These genetic findings, coupled with existing knowledge of vascular histology and physiology, seemed to indicate that mutations in the elastin gene cause this inherited obstructive vascular disorder. The hypothesis has been proven by demonstration of dozens of deletions of the elastin gene in patients with SVAS. To date, no other gene has been associated with a clinical finding of SVAS in its isolated form [16].
Since SVAS is a common feature of Williams syndrome, we hypothesized that mutations involving the elastin gene might also be responsible for this disorder. Mutational analyses of familial and sporadic cases indicated that WS was indeed associated with submicroscopic deletions of chromosome 7q11.23; inherited or de novo constitutional deletion of one elastin allele was identified in every WS patient studied, indicating that hemizygosity (one normal allele, one mutant allele) at this locus is the mechanism of vascular and connective tissue pathology [17]. Taken together, these findings suggested that a developmental reduction in elastin can lead to vascular obstruction, hypertension, premature aging of skin, and other connective tissue abnormalities. We used these data to develop a simple and accurate diagnostic test for WS consisting of fluorescence in situ hybridization (FISH) of metaphase chromosomes with DNA probes specific for the elastin locus. This test is now readily available in most major medical centers.
Since the genomic deletions in WS patients are rarely, if ever, large enough to be detected by cytogenetic methods (i.e., by microscopic evaluation of metaphase chromosomes), any technique for identifying carriers of mutant alleles would require detection at the molecular level. FISH has become a widely invoked method for physically mapping fluorescently tagged genomic DNA fragments (probes) to specific locations on metaphase chromosomes. We adapted this rationale to our own purposes, once we knew that in WS patients the elastin gene should be missing from one homologue of chromosome 7. In nearly all cases, the green fluorescent signal was evident on only one homologue of chromosome 7; the positive control, a probe specific for the pericentromeric region of chromosome 7 that had been labeled with a red fluorophore, was always present on both homologues. When we applied this version of FISH to an evaluation of more than 200 patients, the test was positive in 96% of those who exhibited the clinical criteria for classic WS [18].
The pathogenic mechanisms of vascular obstruction underlying SVAS and WS are not yet understood. Involved may be increased hemodynamic damage to the endothelium of inelastic vessels, causing proliferation of intimal smooth muscle cells and fibroblasts and secondary fibrosis. This hypothesis is supported by clinical reports of improvement of pulmonary vascular stenoses among affected infants treated by postnatal reduction in pulmonary artery pressures. By contrast, obstruction of the aorta and systemic vessels often progresses over time. These changes are coincident with sustained increases in systolic blood pressure after birth.
However, the features of WS other than SVAS are not readily explained by hemizygosity at the elastin locus. Since our data indicate that all WS-associated deletions extend well beyond the elastin gene itself, it is likely that additional, as yet undefined, genes account for the other features of this disorder. Thus, WS is likely to be a contiguous gene deletion syndrome, a syndrome produced (caused) by deletions or mutations in not only the elastin gene but also in adjacent genes of the locus 7q11.23. Molecular characterization of the genomic region surrounding the elastin gene will clarify the basis for such a hypothesis. But even if the additional gene being sought is in fact contiguous as we expect, and can be identified as a likely candidate for some of the manifestations of the WS phenotype, it will be hard to prove its responsibility for specific non-SVAS symptoms because of the difficulty of identifying individuals who may have mutations in that gene but whose elastin homologues are both normal. One approach to this dilemma may be to breed strains of mice with targeted ‘knockouts’ of the candidate gene alone, and to determine ‘clinical’ consequences in this animal model. The exercise, however complicated, could be worth the effort because genes associated with learning disabilities and personality are of great interest to medical science.
Time for primary review 21 days.
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Acknowledgements |
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The author greatly appreciates the editorial assistance of Ruth B. Foltz.
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  U. DeSilva, H. Massa, B. J. Trask, and E. D. Green Comparative Mapping of the Region of Human Chromosome 7 Deleted in Williams Syndrome Genome Res., May 1, 1999; 9(5): 428 - 436. [Abstract] [Full Text]
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