Loss-of-function mutations in the X-linked biglycan gene cause a severe syndromic form of thoracic aortic aneurysms and dissections.

Thoracic aortic aneurysm and dissection (TAAD) is typically inherited in an autosomal dominant manner, but rare X-linked families have been described. So far, the only known X-linked gene is FLNA, which is associated with the periventricular nodular heterotopia type of Ehlers-Danlos syndrome. However, mutations in this gene explain only a small number of X-linked TAAD families. We performed targeted resequencing of 368 candidate genes in a cohort of 11 molecularly unexplained Marfan probands. Subsequently, Sanger sequencing of BGN in 360 male and 155 female molecularly unexplained TAAD probands was performed. We found five individuals with loss-of-function mutations in BGN encoding the small leucine-rich proteoglycan biglycan. The clinical phenotype is characterized by early-onset aortic aneurysm and dissection. Other recurrent findings include hypertelorism, pectus deformity, joint hypermobility, contractures, and mild skeletal dysplasia. Fluorescent staining revealed an increase in TGF-β signaling, evidenced by an increase in nuclear pSMAD2 in the aortic wall. Our results are in line with those of prior reports demonstrating that Bgn-deficient male BALB/cA mice die from aortic rupture. In conclusion, BGN gene defects in humans cause an X-linked syndromic form of severe TAAD that is associated with preservation of elastic fibers and increased TGF-β signaling.Genet Med 19 4, 386-395

Large-scale copy number analysis reveals variations in genes not previously associated with malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is often causally associated with asbestos exposure. Comparative genomic hybridization techniques and arrays demonstrated a complex set of copy number variations (CNVs) in the MPM-genome. These techniques however have a limited resolution, throughput and flexibility compared to next-generation sequencing platforms. In this study, the presence of CNVs in the MPM-genome was investigated using an MPM-cohort (N = 85) for which genomic microarray data are available through 'The Cancer Genome Atlas' (TCGA). To validate these results, the genomes of MPMs and matched normal samples (N = 21) were analyzed using low-pass whole genome sequencing on an 'Illumina HiSeq' platform. CNVs were detected using in-house developed analysis pipelines and frequencies of copy number loss and gain were calculated. In both datasets, losses on chromosomes 1, 3, 4, 6, 9, 13 and 22 and gains on chromosomes 1, 5, 7 and 17 were found in at least 25% and 15% of MPMs, respectively. Besides the well-known MPM-associated genes, CDKN2A, NF2 and BAP1, other interesting cancer-associated genes were listed as frequently involved in a copy number loss (e.g. EP300, SETD2 and PBRM1). Moreover, four cancer-associated genes showed a high frequency of copy number gain in

FRA2A is a CGG repeat expansion associated with silencing of AFF3

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5-12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship

Candidate gene resequencing in a large bicuspid aortic valve-associated thoracic aortic aneurysm cohort: SMAD6 as an important contributor

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter = 4.0 cm in adults, or a Z-score = 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype

Heimler Syndrome is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities and occasional or late onset retinal pigmentation. We ascertained eight families with HS, and - using a whole exome sequencing approach - identified biallelic mutations in PEX1 or PEX6 in six of them. Loss of function mutations in both genes are known causes of a spectrum of autosomal recessive peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the overlap is minimal and the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define Heimler syndrome as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6

Novel microdeletions on chromosome 14q32.2 suggest a potential role for non-coding RNAs in Kagami-Ogata syndrome

In approximately 20% of individuals with Kagami-Ogata syndrome (KOS14, MIM 608149), characterized by a bell-shaped thorax with coat-hanger configuration of the ribs, joint contractures, abdominal wall defects and polyhydramnios during the pregnancy, the syndrome is caused by a maternal deletion of the imprinted gene cluster in chromosome 14q32.2. Most deletions reported so far included one or both of the differentially methylated regions (DMRs) – DLK1/MEG3 IG-DMR and MEG3-DMR. We present two unrelated families with two affected siblings each, presenting with classical KOS14 due to maternally inherited microdeletions. Interestingly, all four patients have lived through to adulthood, even though mortality rates for patients with KOS14 due to a microdeletion are relatively high. In the first family, none of the DMRs is included in the deletion and the methylation status is identical to that of controls. Deletions that do not encompass the DMRs in this region are thus sufficient to elicit the full KOS14 phenotype. In the second family, a partially overlapping deletion including both DMRs and MEG3 was detected. In summary, we show that patients with KOS14 can live into adulthood, that causal deletions do not have to include the DMRs and that consequently a normal methylation pattern does not exclude KOS14

Novel microdeletions on chromosome 14q32.2 suggest a potential role for non-coding RNAs in Kagami-Ogata syndrome

In approximately 20% of individuals with Kagami-Ogata syndrome (KOS14, MIM 608149), characterized by a bell-shaped thorax with coat-hanger configuration of the ribs, joint contractures, abdominal wall defects and polyhydramnios during the pregnancy, the syndrome is caused by a maternal deletion of the imprinted gene cluster in chromosome 14q32.2. Most deletions reported so far included one or both of the differentially methylated regions (DMRs) – DLK1/MEG3 IG-DMR and MEG3-DMR. We present two unrelated families with two affected siblings each, presenting with classical KOS14 due to maternally inherited microdeletions. Interestingly, all four patients have lived through to adulthood, even though mortality rates for patients with KOS14 due to a microdeletion are relatively high. In the first family, none of the DMRs is included in the deletion and the methylation status is identical to that of controls. Deletions that do not encompass the DMRs in this region are thus sufficient to elicit the full KOS14 phenotype. In the second family, a partially overlapping deletion including both DMRs and MEG3 was detected. In summary, we show that patients with KOS14 can live into adulthood, that causal deletions do not have to include the DMRs and that consequently a normal methylation pattern does not exclude KOS14