De Novo Mutations in the BMPR2 Gene in Patients with Heritable Pulmonary Arterial Hypertension

A substantial proportion of patients with pulmonary arterial hypertension (PAH) have mutations in the Bone Morphogenetic Protein Receptor type‐2 (BMPR2) gene. PAH due to BMPR2 mutations is inherited as an autosomal dominant trait with several unique features, including a wide variety of mutations, reduced penetrance, a skewed gender ratio, variable expressivity and genetic anticipation. To address the genetic background of these unique features of BMPR2 mutation, we conducted a systematic analysis of 15 PAH families with BMPR2 mutation. The exonic protein coding sequence of BMPR2 was amplified by polymerase chain reaction and the products were sequenced directly to detect point mutations in BMPR2. Parental identification was carried out to confirm the parental relationship using multiplex 15 loci analysis. Combining mutation detection in family members with parental identification, we described three cases of de novo mutation in the BMPR2 gene by different modes in a PAH family. These de novo mutations may account for the wide variety of mutations in BMPR2. Taken together with the juvenile onset of the disease, there is possibly some balance of de novo mutations and untransmittable mutations which keeps the frequency of PAH low in the general population.     

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II‐like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next‐generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus‐specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.     

Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Somatic alterations in juvenile polyps from BMPR1A and SMAD4 mutation carriers

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder predisposing to gastrointestinal hamartomatous polyps and cancer with a pathogenic SMAD4 or BMPR1A germline mutation (1st‐hit) being identified in about 40–50% of patients. Little is known, however, about the occurrence and nature of somatic alterations (2nd‐hit) in SMAD4‐/BMPR1A‐related juvenile polyps. In this study, we screened 25 polyps from three patients carrying either a pathogenic SMAD4 (c.1244‐1247delACAG) or BMPR1A (c.583C>T; p.Gln195*) germline mutation for somatic alterations. The SMAD4‐related polyps were also analyzed for SMAD4 protein expression by immunohistochemistry. Despite comprehensive screening for loss of heterozygosity (LOH), mutations in the coding sequence, chromosomal rearrangements, and promoter methylation, no somatic alterations could be identified in 14 SMAD4‐related polyps. SMAD4 protein expression, however, was lost in 8 (57%) of 14 juvenile polyps with 6 showing concomitant loss in both, the epithelial and stromal, compartments. In the BMPR1A‐related polyps, five out of nine (56%) displayed LOH. Further analysis of selected polyps revealed that LOH was gene copy number neutral and had occurred in the epithelial compartment. The heterogeneity of genetic mutations and protein expression levels indicates that different modes of gene inactivation can be operational in SMAD4 ‐ and BMPR1A‐related polyp formation. Our observation, that about half of BMPR1A‐related polyps displayed LOH, predominantly in the epithelial compartment, is compatible with BMPR1A acting as a tumour suppressor gene. Still, it remains to be determined whether juvenile polyp development generally requires loss of BMPR1A expression or, as observed in some SMAD4‐related polyps, can occur despite normal protein expression. © 2015 Wiley Periodicals, Inc.     

An homozygous mutation in KCNK3 is associated with an aggressive form of hereditary pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare devastating disease characterized by a high genetic heterogeneity with several related genes recently described, including BMPR2,TBX4 and KCNK3. The association between KCNK3 and PAH has been recently identified, but the prognosis and phenotype associated with these mutations have been poorly described. We studied a series of 136 idiopathic and hereditary PAH Spanish patients for BMPR2, TBX4 and KCNK3 mutations. We report the results of KCNK3 in which we were able to describe two new mutations (p.Gly106Arg and p.Leu214Arg) in three patients. The first one was found in a patient belonging to a consanguineous Romani family, who carried a homozygous mutation in KCNK3 and developed a severe and early form of the disease. To the best of our knowledge, this is the first time that a homozygous mutation in KCNK3 is reported in a PAH patient. The second one was found in a patient who presented at the young adult age a severe form of the disease. The present report supports the contribution of KCNK3 mutations to the genetic etiology of PAH and strongly suggests that mutations in KCNK3 follow incomplete dominance with worsening of the clinical features in homozygous patients.     

Connectivity map analysis of nonsense-mediated decay-positive BMPR2-related hereditary pulmonary arterial hypertension provides insights into disease penetrance

The molecular mechanisms underlying the reduced penetrance seen in the nonsense-mediated decay-positive (NMD+) BMPR2 mutation-associated hereditary pulmonary arterial hypertension (HPAH) remain unknown. We reasoned that the cellular and genetic mechanisms behind this phenomenon could be uncovered by combining expression profiling with Connectivity Map (cMap) analysis. Cultured lymphocytes from 10 patients with HPAH and 10 matched familial control subjects, all with NMD+ BMPR2 mutations, were subjected to expression analysis. For each group, the expression data were combined before analysis. This generated a signature of 23 up-regulated and 12 down-regulated genes in patients with HPAH compared with control subjects (the "PAH penetrance signature"). Although gene set enrichment analysis of this signature was not uniquely informative, cMap analysis identified drugs with expression signatures similar to the PAH penetrance signature. Several of these drugs were predicted to influence reactive oxygen species (ROS) formation. This hypothesis was tested and confirmed in the same cells initially subjected to the expression analysis using quantitative biochemical detection of ROS concentration. We conclude that expression of the PAH penetrance signature represents an increased risk of developing clinical HPAH and that ROS formation may play a role in pathogenesis of HPAH. These results provide the first molecular insights into NMD+ BMPR2 related HPAH penetrance and highlight the potential utility of cMap analyses in pulmonary research.     

Acute Intermittent Porphyria: Predicted Pathogenicity of HMBS Variants Indicates Extremely Low Penetrance of the Autosomal Dominant Disease

Acute intermittent porphyria results from hydroxymethylbilane synthase (HMBS) mutations that markedly decrease HMBS enzymatic activity. This dominant disease is diagnosed when heterozygotes have life‐threatening acute attacks, while most heterozygotes remain asymptomatic and undiagnosed. Although >400 HMBS mutations have been reported, the prevalence of pathogenic HMBS mutations in genomic/exomic databases, and the actual disease penetrance are unknown. Thus, we interrogated genomic/exomic databases, identified non‐synonymous variants (NSVs) and consensus splice‐site variants (CSSVs) in various demographic/racial groups, and determined the NSV's pathogenicity by prediction algorithms and in vitro expression assays. Caucasians had the most: 58 NSVs and two CSSVs among ～92,000 alleles, a 0.00575 combined allele frequency. In silico algorithms predicted 14 out of 58 NSVs as “likely‐pathogenic.” In vitro expression identified 10 out of 58 NSVs as likely‐pathogenic (seven predicted in silico), which together with two CSSVs had a combined allele frequency of 0.00056. Notably, six presumably pathogenic mutations/NSVs in the Human Gene Mutation Database were benign. Compared with the recent prevalence estimate of symptomatic European heterozygotes (～0.000005), the prevalence of likely‐pathogenic HMBS mutations among Caucasians was >100 times more frequent. Thus, the estimated penetrance of acute attacks was ～1% of heterozygotes with likely‐pathogenic mutations, highlighting the importance of predisposing/protective genes and environmental modifiers that precipitate/prevent the attacks.     

Higher‐than‐expected population prevalence of potentially pathogenic germline TP53 variants in individuals unselected for cancer history

Li–Fraumeni syndrome (LFS) is an autosomal‐dominant cancer predisposition disorder associated with pathogenic germline variants in TP53, with a high penetrance over an individual's lifetime. The actual population prevalence of pathogenic germline TP53 mutations is still unclear, most likely due to biased selection of cancer affected families. The aim of this study was to estimate the population prevalence of potentially pathogenic TP53 exonic variants in three sequencing databases, totaling 63,983 unrelated individuals. Potential pathogenicity was defined using an original algorithm combining bioinformatic prediction tools, suggested clinical significance, and functional data. We identified 34 different potentially pathogenic TP53 variants in 131 out of 63,983 individuals (0.2%). Twenty‐eight (82%) of these variants fell within the DNA‐binding domain of TP53, with an enrichment for specific variants that were not previously identified as LFS mutation hotspots, such as the p.R290H and p.N235S variants. Our findings reveal that the population prevalence of potentially pathogenic TP53 variants may be up to 10 times higher than previously estimated from family‐based studies. These results point to the need for further studies aimed at evaluating cancer penetrance modifiers as well as the risk associated between cancer and rare TP53 variants.     

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298‐2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation‐specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation‐specific treatments for PKU.     

The SCN1A Mutation Database: Updating Information and Analysis of the Relationships among Genotype,Functional Alteration,and Phenotype

Mutations in the SCN1A gene have been identified in epilepsy patients with widely variable phenotypes and modes of inheritance and in asymptomatic carriers. This raises challenges in evaluating the pathogenicity of SCN1A mutations. We systematically reviewed all SCN1A mutations and established a database containing information on functional alterations. In total, 1,257 mutations have been identified, of which 81.8% were not recurrent. There was a negative correlation between phenotype severity and missense mutation frequency. Further analyses suggested close relationships among genotype, functional alteration, and phenotype. Missense mutations located in different sodium channel regions were associated with distinct functional changes. Missense mutations in the pore region were characterized by the complete loss of function, similar to haploinsufficiency. Mutations with severe phenotypes were more frequently located in the pore region, suggesting that functional alterations are critical in evaluating pathogenicity and can be applied to patient management. A negative correlation was found between phenotype severity and familial incidence, and incomplete penetrance was associated with missense and splice site mutations, but not truncations or genomic rearrangements, suggesting clinical genetic counseling applications. Mosaic mutations with a load of 12.5–25.0% were potentially pathogenic with low penetrance, suggesting the need for future studies on less pathogenic genomic variations.     

BMPR2 mutation in a patient with pulmonary arterial hypertension and suspected hereditary hemorrhagic telangiectasia

Pulmonary arterial hypertension (PAH) and hereditary hemorrhagic telangiectasia (HHT) are distinct clinical entities caused by germline mutations in genes encoding members of the TGFbeta/BMP superfamily: BMPR2 in PAH and ACVRL1, ENG, or SMAD4 in HHT. When PAH and HHT occasionally co-exist within the same family, ACVRL1 mutations predominate. We report a 36-year-old woman initially diagnosed with PAH at age 24. At 35, following massive hemoptysis, multiple pulmonary arteriovenous malformations were discovered, prompting evaluation for HHT. She met the Cura?ao diagnostic criteria for suspected HHT based on additional findings of nasal telangiectases and epistaxis. Mutation analysis of ACVRL1, ENG, and SMAD4 was normal, but a germline nonsense mutation in BMPR2 was identified. This is the first known report of HHT features, particularly pulmonary AVMs, associated with a BMPR2 mutation. It adds further weight to a common molecular pathogenesis in PAH and HHT, and highlights that BMPR2 gene analysis is indicated in patients affected with both HHT and PAH.     

Hypoparathyroidism,deafness, and renal dysplasia syndrome: 20 Years after the identification of the first GATA3 mutations

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by heterozygous mutations of the GATA3 gene. In the last 20 years, since the identification of the genetic cause of the HDR syndrome, GATA3 mutations have been reported in 124 families (177 patients). The clinical aspects and molecular genetics of the HDR syndrome are reviewed here together with the reported mutations and phenotypes. Reported mutations consist of 40% frameshift deletions or insertions, 23% missense mutations, 14% nonsense mutations, 6% splice‐site mutations, 1% in‐frame deletions or insertions, 15% whole‐gene deletions, and 1% whole‐gene duplication. Missense mutations were found to cluster in the regions encoding the two GATA3 zinc‐finger domains. Patients showed great clinical variability and the penetrance of each HDR defect increased with age. The most frequently observed abnormality was deafness (93%), followed by hypoparathyroidism (87%) and renal defects (61%). The mean age of diagnosis of HDR was 15.3, 7.5, and 14.0 years, respectively. However, patients with whole‐gene deletions and protein‐truncating mutations were diagnosed earlier than patients with missense mutations.     

A rare homozygous missense GDF2 (BMP9) mutation causing PAH in siblings: Does BMP10 status contribute?

Pulmonary arterial hypertension (PAH) is a disease characterized by pathological remodeling of the pulmonary vasculature causing elevated pulmonary artery pressures and ultimately, right ventricular failure from chronic pressure overload. Heterozygous pathogenic GDF2 (encoding bone morphogenetic protein 9 (BMP9)) variants account for some (>1%) adult PAH cases. Only three pediatric PAH cases, harboring homozygous or compound heterozygous variants, are reported to date. Ultra-rare pathogenic GDF2 variants are reported in hereditary hemorrhagic telangiectasia and overlapping disorders characterized by telangiectasias and arteriovenous malformations (AVMs). Here, we present two siblings with PAH homozygous for a GDF2 mutation that impairs BMP9 proprotein processing and reduces growth factor domain availability. We confirm an absence of measurable plasma BMP9 whereas BMP10 levels are detectable and serum-dependent endothelial BMP activity is evident. This contrasts with the absence of activity which we reported in two children with homozygous pathogenic GDF2 nonsense variants, one with PAH and one with pulmonary AVMs, both with telangiectasias, suggesting loss of BMP10 and endothelial BMP activity in the latter may precipitate telangiectasia development. An absence of phenotype in related heterozygous GDF2 variant carriers suggests incomplete penetrance in PAH and AVM-related diseases, indicating that additional somatic and/or genetic modifiers may be necessary for disease precipitation.     

Two novel disease-causing variants in BMPR1B are associated with brachydactyly type A1

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2–5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.     

A founder EIF2AK4 mutation causes an aggressive form of pulmonary arterial hypertension in Iberian Gypsies

Pulmonary arterial hypertension (PAH) is a pathological condition characterized by a persistent and progressive elevation of pulmonary vascular resistance with devastating consequences if untreated. In the past recent years, several genes have been related to PAH, however, the molecular defect remains unknown in a significant proportion of patients with familial PAH (～20%). During the past few years, we have observed that PAH shows a particular behavior in Iberian Gypsies, with more aggressive course and frequently affecting multiple members of the same family. We studied five Gypsy families in whom at least one individual from each family developed a severe form of PAH and in whom no mutation had been identified in the common genes. We applied SNP‐array‐based homozygosity mapping in three families and obtained, among others, one of which included the gene EIF2AK4, recently reported in patients with PAH from group‐1' pulmonary veno‐occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH). Subsequently, we sequenced EIF2AK4 and found a homozygous mutation in all five families: c.3344C>T(p.P1115L). The majority of our patients required early lung transplantation. Hence, this mutation appeared with a more severe phenotype than previously reported for other EIF2AK4 mutations. The finding of this novel mutation is important for genetic counseling and calculation of population recurrence risks.     

Cdkn2a suppresses metastasis in squamous cell carcinomas induced by the gain‐of‐function mutant p53R172H

p53 (TP53) is the most frequently mutated gene in squamous cell carcinomas (SCCs) of the skin and head and neck. Certain p53 mutations are oncogenic and promote invasion and metastasis in SCCs. However, it is unclear how the oncogenic function of mutant p53 is modulated by other molecular alterations that co‐exist in SCCs. Here, we show that deletion of the p53 gene and activation of an endogenous p53^R172H gain‐of‐function mutation in the skin induce carcinomas with similar kinetics and penetrance. Deletion of p53 induced primarily well‐differentiated SCCs. However, most of the tumours induced by p53^R172H were poorly differentiated SCCs, the only metastatic tumours in this model. These tumours expressed higher levels of cyclin D1 than the well‐differentiated SCCs and spindle carcinomas that developed in these mice. Unexpectedly, metastasis was not observed in mice that developed spindle carcinomas, which expressed high levels of the tumour suppressors p16^Ink4a and p19^Arf, encoded by Cdkn2a, a gene frequently deleted in human SCCs. Remarkably, deletion of the Cdkn2a gene in p53^R172H‐induced SCCs promoted a dramatic increase in metastasis rates and a shorter survival in mice that developed these tumours, compared with those observed in mice with tumours in which Cdkn2a was deleted in the presence of a p53 loss‐of‐function mutation or wild‐type p53. Accordingly, the survival of patients with head and neck SCCs bearing co‐occurring high‐risk p53 mutations and CDKN2A homozygous deletions was much shorter than that of patients with tumours in which high‐risk p53 mutations did not contain CDKN2A homozygous deletions, or that of patients with tumours in which homozygous CDKN2A deletions co‐existed with either low‐risk p53 mutations or potential loss‐of‐function mutations in p53. These findings genetically identify a population of SCC patients with worst outcomes and will help to predict outcomes according to the p53 status and alterations in CDKN2A. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.