Copy-number variation in BMPR2 is not associated with the pathogenesis of pulmonary arterial hypertension

<p>Abstract</p> <p>Background</p> <p>Copy-number variations (CNVs) are structural variations in the genome involving 1 kb to 3 mb of DNA. CNV has been reported within intron 1 of the <it>BMPR2 </it>gene. We propose that CNV could affect phenotype in familial and/or sporadic pulmonary arterial hypertension (PAH) by altering gene expression.</p> <p>Methods</p> <p>97 human DNA samples were obtained which included 24 patients with familial PAH, 18 obligate carriers (<it>BMPR2 </it>mutation positive), 20 sporadic PAH patients, and 35 controls. Two sets of primers were designed within the CNV, and two sets of control primers were designed outside the CNV. Quantitative PCR was performed to quantify genomic copies of CNV and control sequences.</p> <p>Results</p> <p>A CNV in <it>BMPR2 </it>was present in one African American negative control subject.</p> <p>Conclusion</p> <p>We conclude that the CNV in intron 1 in <it>BMPR2 </it>is unlikely to play a role in the pathogenesis of either familial or sporadic PAH.</p> <p>Trial Registration</p> <p>NIH NCT00091546.</p

Gene expression in BMPR2 mutation carriers with and without evidence of Pulmonary Arterial Hypertension suggests pathways relevant to disease penetrance

<p>Abstract</p> <p>Background</p> <p>While BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH), only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Furthermore, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself.</p> <p>Methods</p> <p>To resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses.</p> <p>Results</p> <p>By gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. One of these, CYP1B1, had tenfold lower expression than control groups in female but not male PAH patients. Analysis of overrepresented gene ontology groups suggests that risk of disease correlates with alterations in pathways more strongly than with any specific gene within those pathways.</p> <p>Conclusion</p> <p>Disease status in BMPR2 mutation carriers was correlated with alterations in proliferation, GTP signaling, and stress response pathway expression. The estrogen metabolizing gene CYP1B1 is a strong candidate as a modifier gene in female PAH patients.</p

Telomerase mutations in families with idiopathic pulmonary fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis is progressive and often fatal; causes of familial clustering of the disease are unknown. Germ-line mutations in the genes hTERT and hTR, encoding telomerase reverse transcriptase and telomerase RNA, respectively, cause autosomal dominant dyskeratosis congenita, a rare hereditary disorder associated with premature death from aplastic anemia and pulmonary fibrosis. METHODS: To test the hypothesis that familial idiopathic pulmonary fibrosis may be caused by short telomeres, we screened 73 probands from the Vanderbilt Familial Pulmonary Fibrosis Registry for mutations in hTERT and hTR. RESULTS: Six probands (8%) had heterozygous mutations in hTERT or hTR; mutant telomerase resulted in short telomeres. Asymptomatic subjects with mutant telomerase also had short telomeres, suggesting that they may be at risk for the disease. We did not identify any of the classic features of dyskeratosis congenita in five of the six families. CONCLUSIONS: Mutations in the genes encoding telomerase components can appear as familial idiopathic pulmonary fibrosis. Our findings support the idea that pathways leading to telomere shortening are involved in the pathogenesis of this disease

Ancestral Mutation in Telomerase Causes Defects in Repeat Addition Processivity and Manifests As Familial Pulmonary Fibrosis

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease

Alzheimer's Disease Amyloid-β Links Lens and Brain Pathology in Down Syndrome

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-β peptides (Aβ), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Aβ accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Aβ pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Aβ accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Aβ aggregates (∼5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Aβ in DS lenses. Incubation of synthetic Aβ with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Aβ accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD

Three-dimensional spatial analysis of missense variants in RTEL1 identifies pathogenic variants in patients with Familial Interstitial Pneumonia

Abstract Background Next-generation sequencing of individuals with genetic diseases often detects candidate rare variants in numerous genes, but determining which are causal remains challenging. We hypothesized that the spatial distribution of missense variants in protein structures contains information about function and pathogenicity that can help prioritize variants of unknown significance (VUS) and elucidate the structural mechanisms leading to disease. Results To illustrate this approach in a clinical application, we analyzed 13 candidate missense variants in regulator of telomere elongation helicase 1 (RTEL1) identified in patients with Familial Interstitial Pneumonia (FIP). We curated pathogenic and neutral RTEL1 variants from the literature and public databases. We then used homology modeling to construct a 3D structural model of RTEL1 and mapped known variants into this structure. We next developed a pathogenicity prediction algorithm based on proximity to known disease causing and neutral variants and evaluated its performance with leave-one-out cross-validation. We further validated our predictions with segregation analyses, telomere lengths, and mutagenesis data from the homologous XPD protein. Our algorithm for classifying RTEL1 VUS based on spatial proximity to pathogenic and neutral variation accurately distinguished 7 known pathogenic from 29 neutral variants (ROC AUC = 0.85) in the N-terminal domains of RTEL1. Pathogenic proximity scores were also significantly correlated with effects on ATPase activity (Pearson r = −0.65, p = 0.0004) in XPD, a related helicase. Applying the algorithm to 13 VUS identified from sequencing of RTEL1 from patients predicted five out of six disease-segregating VUS to be pathogenic. We provide structural hypotheses regarding how these mutations may disrupt RTEL1 ATPase and helicase function. Conclusions Spatial analysis of missense variation accurately classified candidate VUS in RTEL1 and suggests how such variants cause disease. Incorporating spatial proximity analyses into other pathogenicity prediction tools may improve accuracy for other genes and genetic diseases