Characterization of large genomic deletions in the FBN1 gene using multiplex ligation-dependent probe amplification

<p>Abstract</p> <p>Background</p> <p>Connective tissue diseases characterized by aortic aneurysm, such as Marfan syndrome, Loeys-Dietz syndrome and Ehlers Danlos syndrome type IV are heterogeneous and despite overlapping phenotypes, the natural history, clinical manifestations and interventional course for each diagnosis can be quite unique. The majority of mutations involved in the etiology of these disorders are missense and nonsense mutations. However, large deletions and duplications undetected by sequencing may be implicated in their pathogenesis, and may explain the apparent lack of genotype-phenotype correlation in a subset of patients. The objective of this study was to search for large pathogenic deletions and/or duplications in the <it>FBN1, TGFβR1</it>, and <it>TGFβR2 </it>genes using multiplex-ligation dependent probe amplification (MLPA) in patients with aortopathy, in whom no mutations in the <it>FBN1, TGFβR1</it>, and <it>TGFβR2 </it>genes were identified by sequencing.</p> <p>Methods</p> <p>The study included 14 patients from 11 unrelated families with aortic aneurysm. Of those, six patients (including 3 first-degree relatives), fulfilled the revised Ghent criteria for Marfan syndrome, and eight had predominantly aortic aneurysm/dilatation with variable skeletal and craniofacial involvement. MLPA for <it>FBN1, TGFβR1</it>, and <it>TGFβR2 </it>was carried out in all patients. A 385 K chromosome 15 specific array was used in two patients with a deletion of the entire <it>FBN1 </it>in order to define its size and boundaries.</p> <p>Results</p> <p>We identified two novel large deletions in the <it>FBN1 </it>gene in four patients of two unrelated families who met clinical diagnostic criteria for Marfan syndrome. One patient was found to have a <it>FBN1 </it>deletion encompassing exons 1-5. The other three patients had a 542 Kb deletion spanning the whole <it>FBN1 </it>gene and five additional genes (<it>SLC24A5, MYEF2, CTXN2, SLC12A1, DUT</it>) in the chromosome 15.</p> <p>Conclusions</p> <p>Our findings expand the number of large <it>FBN1 </it>deletions, and emphasize the importance of screening for large genomic deletions in connective tissue disorders featuring aortopathies, especially for those with classic Marfan phenotype.</p

Parkes Weber syndrome associated with two somatic pathogenic variants in RASA1

Parkes Weber syndrome is associated with autosomal dominant inheritance, caused by germline heterozygous inactivating changes in th

5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia

<p>Abstract</p> <p>Background</p> <p>Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (<it>ACVRL1</it>) or Endoglin (<it>ENG</it>) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of <it>ENG</it>.</p> <p>Methods and Results</p> <p>We sequenced the 5'UTR of <it>ENG </it>for 154 HHT patients without mutations in <it>ENG </it>or <it>ACVRL1 </it>coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the <it>ENG</it>, as well as in three other patients, one of which had an affected sibling with the same mutation. <it>In vitro </it>expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation.</p> <p>Conclusions</p> <p>Our results emphasize the need for the inclusion of the 5'UTR region of <it>ENG </it>in clinical testing for HHT.</p

BMP9 Mutations Cause a Vascular-Anomaly Syndrome with Phenotypic Overlap with Hereditary Hemorrhagic Telangiectasia

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-β) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT

Homozygous GDF2 nonsense mutations result in a loss of circulating BMP9 and BMP10 and are associated with either PAH or an "HHT-like" syndrome in children.

BACKGROUND: Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra-rare homozygous GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH (c.[76C>T];[76C>T] or p.[Gln26Ter];[Gln26Ter] and a new individual with pulmonary arteriovenous malformations (PAVMs; c.[835G>T];[835G>T] or p.[Glu279Ter];[Glu279Ter]); both with facial telangiectases. METHODS: Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE-luciferase reporter cell line (HMEC1-BRE). Proteins were expressed for assessment of secretion and processing. RESULTS: Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum-derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9. CONCLUSION: Collectively, these data show that homozygous GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or "HHT-like" telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs

Characterization of a family mutation in the 5' untranslated region of the endoglin gene causative of hereditary hemorrhagic telangiectasia

7 p.-4 fig.Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease characterized by nose and gastrointestinal bleeding, telangiectases in skin and mucosa, and arteriovenous malformations in major internal organs. Most patients carry a mutation in the coding region of the endoglin (ENG) or activin A receptor type II-1 (ACVRL1) gene. Nonetheless, in around 15% of patients, sequencing analysis and duplication/deletion tests fail to pinpoint mutations in the coding regions of these genes. In these cases, it has been shown that sequencing of the 5'-untranslated region (5'UTR) of ENG may be useful to identify novel mutations in the ENG non-coding region. Here we report the genetic characterization and functional analysis of the heterozygous mutation c.-142A>T in the 5'UTR region of ENG found in a family with several members affected by HHT. This variant gives rise to a new initiation codon of the protein that involves the change in its open reading frame. Transfection studies in monkey cells using endoglin expression vectors demonstrated that c-142A>T mutation results in a clear reduction in the levels of the endoglin protein. These results support the inclusion of the 5'UTR of ENG in the standard genetic testing for HHT to increase its sensitivity.This work was supported by grants from Ministerio de Economia, Industria y Competitividad (SAF2013-43421-R to CB), Mobility Program Salvador de Madariaga, Ministerio de Educacion, Cultura y Deporte (PRX17/00142 to CB), Consejo Superior de Investigaciones Cientificas (201420E039 and 201920E022 to CB), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to CB and contract CNV-234-PRF-360 to LR-L) of Spain.Peer reviewe

Genetic Variants Associated with Port-Wine Stains

Background: Port-wine stains (PWS) are capillary malformations, typically located in the dermis of the head and neck, affecting 0.3% of the population. Current theories suggest that port-wine stains are caused by somatic mutations that disrupt vascular development. Objectives: Understanding PWS genetic determinants could provide insight into new treatments. Methods: Our study used a custom next generation sequencing (NGS) panel and digital polymerase chain reaction to investigate genetic variants in 12 individuals with isolated port-wine stains. Importantly, affected and healthy skin tissue from the same individual were compared. A subtractive correction method was developed to eliminate background noise from NGS data. This allowed the detection of a very low level of mosaicism. Results: A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was found in one case with 4% allele frequency. The previously reported GNAQ c.548G>A, p.Arg183Gln was confirmed in 9 of 12 cases with an allele frequency ranging from 1.73 to 7.42%. Digital polymerase chain reaction confirmed novel variants detected by next generation sequencing. Two novel somatic variants were also found in RASA1, although neither was predicted to be deleterious. Conclusions: This is the second largest study on isolated, non-syndromic PWS. Our data suggest that GNAQ is the main genetic determinant in this condition. Moreover, isolated port-wine stains are distinct from capillary malformations seen in RASA1 disorders, which will be helpful in clinical evaluation