Skin elastic fibers in Williams syndrome

The elastin gene is consistently deleted in Williams syndrome and as this protein represents the major component of the elastic fibers of the dermis, we sought to investigate skin elastic fibers in Williams syndrome as a key to unraveling extracellular matrix disorganization in this condition. Both morphometric parameters analyzed by using automated image analysis and immunofluorescence labeling with monoclonal antibodies against elastin and fibrillin 1 showed a disorganized pre-elastic (oxytalan and elaunin) and mature elastic fibers in the dermis of 10 Williams syndrome patients compared with five healthy children and one patient with isolated supravalvular aortic stenosis. Skin biopsies in Williams syndrome patients provide a simple mean to elucidate extracellular matrix anomalies. Hopefully, this method could give clues to the understanding of the elastic network anomalies in this condition and even to the consequences of these latter on elasticity and resilience of other tissues such as the arterial tree. Am. J. Med. Genet. 87:134–138, 1999. © 1999 Wiley-Liss, Inc.     

Delineation of the common critical region in Williams syndrome and clinical correlation of growth,heart defects,ethnicity, and parental origin

Williams syndrome (WS) is a neurodevelopmental disorder with a variable phenotype. Molecular genetic studies have indicated that hemizygosity at the elastin locus (ELN) may account for the cardiac abnormalities seen in WS, but that mental retardation and hypercalcemia are likely caused by other genes flanking ELN. In this study, we defined the minimal critical deletion region in 63 patients using 10 microsatellite markers and 5 fluorescence in situ hybridization (FISH) probes on chromosome 7q, flanking ELN. The haplotype analyses showed the deleted cases to have deletions of consistent size, as did the FISH analyses using genomic probes for the known ends of the commonly deleted region defined by the satellite markers. In all informative cases deleted at ELN, the deletion extends from D7S489U to D7S1870. The genetic distance between these two markers is about 2 cM. Of the 51 informative patients with deletions, 29 were maternal and 22 were paternal in origin. There was no evidence for effects on stature by examining gender, ethnicity, cardiac status, or parental origin of the deletion. Heteroduplex analysis for LIMK1, a candidate gene previously implicated in the WS phenotype, did not show any mutations in our WS patients not deleted for ELN. LIMK1 deletions were found in all elastin-deletion cases who had WS. One case, who has isolated, supravalvular aortic stenosis and an elastin deletion, was not deleted for LIMK1. It remains to be determined if haploinsufficiency of LIMK1 is responsible in part for the WS phenotype or is simply deleted due to its close proximity to the elastin locus. Am. J. Med. Genet. 78:82–89, 1998. © 1998 Wiley-Liss, Inc.     

Spectrum of elastin sequence variants and cardiovascular phenotypes in 49 patients with Williams–Beuren syndrome

Haploinsufficiency of the elastin gene (ELN) on 7q11.23 is responsible for supravalvular aortic stenosis (SVAS) and other arteriopathies in patients with Williams–Beuren syndrome (WBS). These defects occur with variable penetrance and expressivity, but the basis of this is unknown. To determine whether DNA variations in ELN could serve as genetic modifiers, we sequenced the 33 exons and immediately surrounding sequence of the ELN gene (9,455 bp of sequence) in 49 DNAs from patients with WBS and compared cardiovascular phenotypes. Four missense, and four novel intronic variants were identified from a total of 24 mostly intronic single nucleotide variations and one indel. Two missense changes were present in one patient each, one published, p.Gly610Ser in exon 27 (MAF, 0.003) and one novel, p.Cys714Tyr, in exon 33 (MAF, 0.001), were rare in the general population. To identify a statistical association between the variants identified here and cardiovascular phenotypes a larger cohort would be needed. © 2013 Wiley Periodicals, Inc.     

Molecular and clinical correlation study of Williams-Beuren syndrome: No evidence of molecular factors in the deletion region or imprinting affecting clinical outcome

Williams-Beuren syndrome (WBS) results from a deletion of 7q11.23 in 90–95% of all clinically typical cases. Clinical manifestation can be variable and therefore, deletion size, inherited elastin (ELN) and LIM kinase 1 (LIMK1) alleles, gender, and parental origin of deletion have been investigated for associations with clinical outcome. In an analysis of 85 confirmed deletion cases, no statistically significant associations were found after Bonferroni's correction for multiple pairwise comparisons. Furthermore, the present data do not support presence of imprinted genes in the WBS common deletion despite a nonsignificant excess of maternal over paternal deletions. Maternal deletion cases were more likely to have a large head circumference in the present data. Also, pairwise comparisons between individual WBS clinical features have been conducted and revealed significant associations between (1) low birth weight and poor postnatal weight gain (<10th percentile at the time of examination) and (2) transient infantile hypercalcemia and a stellate iris pattern. The latter association could indicate a common underlying etiology. Am. J. Med. Genet. 86:34–43, 1999. © 1999 Wiley-Liss, Inc.     

Supravalvular aortic stenosis cosegregates with a familial 6;7 translocation which disrupts the elastin gene

Supravalvular aortic stenosis (SVAS) is an autosomal dominant disorder characterized by abnormalities of development of the great vessels. SVAS is also commonly part of Williams syndrome. Linkage to the elastin gene on chromosome 7q11 has recently been reported in two kindreds with SVAS. Previous reports of patients with 7q11 deletions have noted great vessel abnormalities in some. We report on a family in which SVAS is cosegregating with a balanced reciprocal translocation, t(6:7) (p21.1;q11.23), providing further evidence that SVAS is the result of mutation of elestin at 7q11.23 region. The propositus of the translocation family has some minor anomalies which occur in Williams syndrome, Suggesting that elestin abnormalities may cause some of the abnormalities found in Williams syndrome. © 1993 Wiley-Liss, Inc. © 1993 Wiley-Liss, Inc.     

Three decades of follow-up of aortic and pulmonary vascular lesions in the Williams-Beuren syndrome

The diagnostic criteria of the Williams-Beuren syndrome (WBS) were established almost 3 decades ago. Until now there has been little knowledge about the natural and post-surgical history of vascular lesions in this syndrome. In order to evaluate the long term follow-up of aortic and pulmonary vascular lesions, we have analysed the catheterization data, angiocardiograms, and Doppler-echo measurements in 59 patients who were seen at least twice in our institution between 1961 and 1993. Their follow-up periods ranged from 2.1 to 28.2 years. Of 45 patients with supravalvular aortic stenosis (SVAS) with a mean follow-up period of 12.9 years, it became evident that pressure gradients of less than 20 mm Hg in infancy generally remained unchanged during the first two decades of life. Pressure gradients exceeding 20 mm Hg increased from an average of 35.5 mm Hg to 52.7 mm Hg in 13 patients. Of these, 8 required surgical relief of the narrowing. In 7 patients aortic hypoplasia was documented. In 5 of them the caliber of the aorta showed a tendency towards normalisation within a period of 11.9 to 23.9 years. Of 6 individuals with aortic hypoplasia and surgical relief of SVAS, 4 patients developed restenosis at the distal end of the aortoplasty patch. In contrast, 9 patients with operated SVAS—but without aortic hypoplasia—remained free of restenosis over a period of 11 years (mean). Coarctation occurred in 4/59 patients; restenosis was seen in 2 after 5 and 16 years. Peripheral pulmonary stenosis was followed in 23 patients over 14.4 years (mean). During this period the systolic pressure gradients fell from 23 to 9.3 mm Hg (mean). In adolescence and adulthood the gradients were below 20 mm Hg in 22/23 individuals. In WBS there is a good long-term prognosis for SVAS if gradients during infancy are low. SVAS with gradients above 20 mm Hg tend to increase; 60% of them require surgical relief with good long-term results. But aortic hypoplasia impairs the prognosis of operated SVAS, because restenosis may occur. Peripheral pulmonary stenosis generally shows a good long-term prognosis. © 1994 Wiley-Liss, Inc.     

Analysis of CBP (CREBBP) gene deletions in Rubinstein-Taybi syndrome patients using real-time quantitative PCR

Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome characterized by facial abnormalities, broad thumbs, broad big toes, and growth and mental retardation as the main clinical features. RTS was shown to be associated with disruption of the CREB-binding protein gene CBP (CREBBP), either by gross chromosomal rearrangements or by point mutations. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (about 10%). Most deletion studies in RTS are performed by FISH analysis, and five cosmids must be used to cover the whole of the CBP gene, which spreads over 150 kb. Here we report the design of gene dosage assays by real-time quantitative PCR that are targeted on three exons located respectively at the 5' end (exon 2), in the middle (exon 12), and at the 3' end (exon 30) of the CBP gene. This technique proved to be efficient and powerful in finding deletions and complementary to the other available techniques, since it allowed us to identify deletions at the 3' end of the gene that had been missed by FISH analysis, and to refine some deletion breakpoints. Our results therefore suggest that real-time quantitative PCR is a useful technique to be included in the deletion search in RTS patients.     

Concurrence of supravalvular aortic stenosis and peripheral pulmonary stenosis in three generations of a family: A form of arterial dysplasia

Isolated supravalvular aortic stenosis (SVAS) commonly is an autosomal dominant trait; it may also occur in the Williams syndrome (WS). While peripheral pulmonary stenosis (PPS) can occur in the same individual with familial isolated SVAS, concurrence of these lesions in different relatives of a family is uncommon. We describe five affected individuals in one family; three had isolated SVAS, one had isolated PPS, and one had SVAS and PPS. Based on this family and review of literature, we suggest that SVAS is a form of arterial dysplasia encompassing PPS in its spectrum. It is developmentally distinct from other left heart obstructive lesions that are hypothesized to be related to blood flow abnormalities in the developing embryo. We also conclude that the clinical disorder in this family represents one that is distinct from WS. © 1993 Wiley-Liss, Inc.     

De Barsy syndrome: Report of a case,literature review,and elastin gene expression studies of the skin

Several “progeroid” syndromes have now been identified. The De Barsy syndrome is an autosomal recessive syndrome of dwarfism, mental deficiency, an “aged” appearance at birth, abnormal elastic fibers on skin biopsy, and lax skin, large helices, eye abnormalities, lax joints, hypotonia, and athetoid posturing. We report one case and review 11 cases from the literature. To understand the abnormal appearance of the elastic fibers on biopsy, we performed elastin gene expression studies on fibroblasts cultured from our patient's skin. Molecular hybridization studies revealed reduced elastin mRNA steady-state levels as compared with age matched control individuals. Assuming normal rates of mRNA translation, reduced elastin synthesis would occur. Diminished dermal elastin content could explain the altered cutaneous elasticity, decreased elastic fibers in the skin, and many clinical manifestations of individuals with this condition.     

Unequal interchromosomal rearrangements may result in elastin gene deletions causing the Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is generally the consequence of an interstitial microdeletion at 7q11.23, which includes the elastin gene, thus causing hemizygosity at the elastin gene locus. The origin of the deletion has been reported by many authors to be maternal in approximately 60% and paternal in 40% of cases. Segregation analysis of grandparental markers flanking the microdeletion region in WBS patients and their parents indicated that in the majority of cases a recombination between grandmaternal and grandpaternal chromosomes 7 at the site of the deletion had occurred during meiosis in the parent from whom the deleted chromosome stemmed. Thus, the majority of deletions were considered a consequence of unequal crossing-over between homologous chromosomes 7 (interchromosomal rearrangement) while in the remaining cases an intrachromosomal recombination (between the chromatids of one chromosome 7) may have occurred. These results suggest that the majority of interstitial deletions of the elastin gene region occur during meiosis, due to unbalanced recombination while a minority could occur before or during meiosis probably due to intrachromosomal rearrangements. The recurrence risk of the interchromosomal rearrangements for sibs of a proband with non-affected parents must be negligible, which fits well with the observation of sporadic occurrence of almost all cases of WBS.      

多重聚合酶链反应检测DMD／BMD患者的基因缺失

目的了解Duchenne型肌营养不良（DMD）／Becker型肌营养不良（BMD）患者基因缺失的分布规律,并探索检测技术。方法应用28对引物多重聚合酶链反应（mPCR）分4组对20例DMD／BMD患者进行Dysophin基因缺失检测分析。结果12例（60％）存在外显子缺失,8例（40％）未检测到缺失;8例缺失片段集中于44—52号外显子,4例集中于5’端外显子。结论基因缺失为主要突变类型,缺失片段主要分布于44—52、2～20号外显子两个缺失热区。     

Detection of hemizygosity in Hermansky-Pudlak syndrome by quantitative real-time PCR

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding diathesis and, in some patients, pulmonary fibrosis or granulomatous colitis. HPS is associated with biosynthesis defects of melanosomes, platelet-dense bodies, and lysosomes. There are seven genetic HPS subtypes; HPS-1 is the most common. We used a real-time quantitative PCR (qPCR) approach to investigate six HPS-1 patients, previously assigned as having homozygous mutations in the HPS1 gene. HPS1 gene copy numbers, calculated by use of a comparative Ct method, revealed that one patient was in fact hemizygous for her c.1189delC (S396delC) HPS1 mutation. The causative deletion/insertion was 13,966 bp in size, with defined breakpoints, and involved an adjacent gene (C10orf33). A mechanism of formation is proposed for the deletion/insertion, and both multiplex and qPCR indicated that the deletion/insertion was present in the patient, her brother, and her father. qPCR amplification is valuable for detecting deletions too small to be identified by fluorescence in situ hybridization. This demonstration of hemizygosity, performed using genomic DNA, can eliminate concerns about non-paternity and can verify the diagnosis of an autosomal recessive disorder when a DNA alteration appears to be homozygous by standard PCR and sequencing methods, and its pathogenicity is in doubt.     

PCR detection of Y-specific sequences in patients with Ullrich-Turner syndrome: Clinical implications and limitations

Cytogenetic analysis of patients with Ullrich-Turner syndrome (UTS) may fail to detect low levels of Y chromosome mosaicism or Y-derived marker chromosomes. More sensitive polymerase chain reaction (PCR)-based tests have been developed; however, applicability of these data to prognosis of virilization and gonadoblastoma development has not been investigated adequately. We used a multiplex PCR-based method to detect two Y-specific sequences, SRY and AMGLY. Thirteen patients with UTS without cytogenetically detected Y chromosomes were studied. Y-specific sequences were detected in 5 patients by multiplex PCR. A cryptic translocation involving the Y chromosome was found in one patient with severe virilization of external genitalia and a male phenotype. Y chromosomal mosaicism was detected in peripheral blood and in both gonads of one patient, and only in the left gonad of another patient. Existence of a Y-derived marker was demonstrated in 2 patients, one of whom had no testicular tissue or virilization. Consistent with previous reports, we conclude that PCR is more sensitive than classical cytogenetic analysis and detects patients with Y-specific sequences in blood cells. However, the absence of Y-specific material in blood is not a sufficient reason to reject surgical treatment in case of virilization. Am. J. Med. Genet. 76:283–287, 1998. © 1998 Wiley-Liss, Inc.     

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.     

Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease

Genetic diagnosis of PLP gene duplications/deletions in patients with Pelizaeus-Merzbacher disease.PMD is an X-linked recessive disorder due to a proteolipid protein (PLP) deficiency. Duplications of PLP gene were shown to be the principle cause of the disorder, accounting for an estimated 50-70% of cases. To define a simple and reliable method for genetic diagnosis of PMD, a group of 42 patients with clinical manifestation of PMD was analyzed by means of real-time quantitative PCR. Parallel fluorescence in situ hybridization (FISH) analysis was performed on the same group of patients. Real-time PCR found seventeen samples had increased gene dosage, whereas FISH detected sixteen duplicated samples. Both methods identified a sample with PLP gene deletion. Our results indicate that real-time PCR is a sensitive and reliable method for the detection of gene duplications/deletions. We further discussed the advantages and limitations of each method in clinical diagnosis of PMD.     

A combined stain for elastin and acid mucopolysaccharides in large blood vessels

A reliable, reproducible combined stain for elastin and acid mucopolysaccharides has been developed and is particularly suitable for the study of large blood vessels as muscle and collagen are also stained. The technique is described.     

Multicolour FISH and quantitative PCR can detect submicroscopic deletions in holoprosencephaly patients with a normal karyotype

Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain. At birth, nearly 50% of children with HPE have cytogenetic anomalies. Approximately 20% of infants with normal chromosomes have sequence mutations in one of the four main HPE genes (SHH, ZIC2, SIX3, and TGIF). The other non‐syndromic forms of HPE may be due to environmental factors or mutations in other genes, or potentially due to submicroscopic deletions of HPE genes. We used two complementary assays to test for HPE associated submicroscopic deletions. Firstly, we developed a multicolour fluorescent in situ hybridisation (FISH) assay using probes for the four major HPE genes and for two candidate genes (DISP1 and FOXA2). We analysed lymphoblastoid cell lines (LCL) from 103 patients who had CNS findings of HPE, normal karyotypes, and no point mutations, and found seven microdeletions. We subsequently applied quantitative PCR to 424 HPE DNA samples, including the 103 samples studied by FISH: 339 with CNS findings of HPE, and 85 with normal CNS and characteristic HPE facial findings. Microdeletions for either SHH, ZIC2, SIX3, or TGIF were found in 16 of the 339 severe HPE cases (that is, with CNS findings; 4.7%). In contrast, no microdeletion was found in the 85 patients at the mildest end of the HPE spectrum. Based on our data, microdeletion testing should be considered as part of an evaluation of holoprosencephaly, especially in severe HPE cases.