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.     

Hereditary hemorrhagic telangiectasia: ENG and ALK-1 mutations in Dutch patients

Hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber disease is an autosomal dominant disorder characterized by an aberrant vascular development. The resulting vascular lesions range from smaller mucocutaneous telangiectases to large visceral arteriovenous malformations, especially in the skin, lung, gastrointestinal tract and the brain. Mutations in the genes encoding endoglin (ENG, chromosome 9q34) and activin A receptor type-like kinase 1 (ALK-1, also named ACVRL1, chromosome 12q13) are associated with HHT1 and HHT2, respectively. We report here on the genetic and molecular heterogeneity found in the HHT population in the Netherlands. Probands of 104 apparently unrelated families were studied and we performed sequence analysis on both the ENG gene and ALK-1 gene. In most of the probands, we found a mutation in one of the two genes: 53% in the ENG gene and 40% in the ALK-1 gene. In 7% of the families no ENG or ALK1 mutation was found. The mutations detected were deletions, insertions, nonsense, missense and splice site mutations. The majority were novel mutations.     

Angiogenesis regulation by TGFβ signalling: clues from an inherited vascular disease

Studies of rare genetic diseases frequently reveal genes that are fundamental to life, and the familial vascular disorder HHT (hereditary haemorrhagic telangiectasia) is no exception. The majority of HHT patients are heterozygous for mutations in either the ENG (endoglin) or the ACVRL1 (activin receptor-like kinase 1) gene. Both genes are essential for angiogenesis during development and mice that are homozygous for mutations in Eng or Acvrl1 die in mid-gestation from vascular defects. Recent development of conditional mouse models in which the Eng or Acvrl1 gene can be depleted in later life have confirmed the importance of both genes in angiogenesis and in the maintenance of a normal vasculature. Endoglin protein is a co-receptor and ACVRL1 is a signalling receptor, both of which are expressed primarily in endothelial cells to regulate TGFβ (transforming growth factor β) signalling in the cardiovasculature. The role of ACVRL1 and endoglin in TGFβ signalling during angiogenesis is now becoming clearer as interactions between these receptors and additional ligands of the TGFβ superfamily, as well as synergistic relationships with other signalling pathways, are being uncovered. The present review aims to place these recent findings into the context of a better understanding of HHT and to summarize recent evidence that confirms the importance of endoglin and ACVRL1 in maintaining normal cardiovascular health.     

Highlights of HRCT imaging in IPF

Background

Aim of this prospective study was to compare clinical and genetic findings in children with idiopathic or heritable pulmonary arterial hypertension (I/HPAH) with children affected with congenital heart defects associated PAH (CHD-APAH).

Methods

Prospectively included were 40 consecutive children with invasively diagnosed I/HPAH or CHD-APAH and 117 relatives. Assessment of family members, pedigree analysis and systematic screening for mutations in TGFß genes were performed.

Results

Five mutations in the bone morphogenetic protein type II receptor (BMPR2) gene, 2 Activin A receptor type II-like kinase-1 (ACVRL1) mutations and one Endoglin (ENG) mutation were found in the 29 I/HPAH children. Two mutations in BMPR2 and one mutation in ACVRL1 and ENG, respectively, are described for the first time. In the 11 children with CHD-APAH one BMPR2 gene mutation and one Endoglin gene mutation were found. Clinical assessment of relatives revealed familial aggregation of the disease in 6 children with PAH (HPAH) and one CHD-APAH patient. Patients with mutations had a significantly lower PVR.

Conclusion

Mutations in different TGFß genes occurred in 8/29 (27.6%) I/HPAH patients and in 2/11 (18.2%) CHD-APAH patients and may influence the clinical status of the disease. Therefore, genetic analysis in children with PAH, especially in those with I/HPAH, may be of clinical relevance and shows the complexity of the genetic background.     

The Contribution of Mosaic Variants to Autism Spectrum Disorder

De novo mutation is highly implicated in autism spectrum disorder (ASD). However, the contribution of post-zygotic mutation to ASD is poorly characterized. We performed both exome sequencing of paired samples and analysis of de novo variants from whole-exome sequencing of 2,388 families. While we find little evidence for tissue-specific mosaic mutation, multi-tissue post-zygotic mutation (i.e. mosaicism) is frequent, with detectable mosaic variation comprising 5.4% of all de novo mutations. We identify three mosaic missense and likely-gene disrupting mutations in genes previously implicated in ASD (KMT2C, NCKAP1, and MYH10) in probands but none in siblings. We find a strong ascertainment bias for mosaic mutations in probands relative to their unaffected siblings (p = 0.003). We build a model of de novo variation incorporating mosaic variants and errors in classification of mosaic status and from this model we estimate that 33% of mosaic mutations in probands contribute to 5.1% of simplex ASD diagnoses (95% credible interval 1.3% to 8.9%). Our results indicate a contributory role for multi-tissue mosaic mutation in some individuals with an ASD diagnosis.     

Bioinformatic analysis of pathogenic missense mutations of activin receptor like kinase 1 ectodomain

Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1(EC)) has been elusive so far. We here describe the building of a homology model for ALK1(EC), followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1(EC) potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1(EC) allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1(EC) and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms.     

Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant bleeding disorder characterized by localized angiodysplasia. Mutations in either of two genes, endoglin or ALK-1, can cause HHT. Both genes encode putative receptors for the transforming growth factor-beta superfamily of ligands. Many mutations in each gene have been identified in HHT kindreds from around the world, and with few exceptions mutations are unique and family specific. The prevalence of HHT in the Leeward Islands of the Netherlands Antilles is possibly the highest of any geographical location. We wished to establish whether this high prevalence is due to a genetic founder effect or to multiple mutational events. HHT kindreds from the Netherlands Antilles and The Netherlands were screened for mutations in the two genes associated with HHT. Haplotype analysis of a 5-cM region on chromosome 9 flanking the endoglin gene revealed three distinct disease haplotypes in the ten Antillean families studied. Seven of these families share a splice-site mutation in exon 1 of endoglin. Two other Antillean families share a missense mutation in exon 9a of endoglin. This mutation was also found in a Dutch family that shares the same disease haplotype as the Antillean families with this mutation. Thus it appears that HHT in the Netherlands Antilles is due to a limited number of ancestral mutations in the endoglin gene, and that one of these mutations was introduced into the African slave population by a Dutch colonist. The limited scope of mutations suggests that a presymptomatic screening program for HHT would be feasible in this population.     

Mutation analysis of the natriuretic peptide precursor B (NPPB) gene in patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetically heterogenous disease caused by mutations in genes that primarily encode sarcomeric proteins. No mutation is identified in up to 40% of HCM patients, suggesting other causative genes exist. Natriuretic peptide precursor B (NPPB; also known as "BNP") is a cardiac hormone involved in body fluid homeostasis and cardiac myocyte growth. NPPB concentrations are markedly increased in patients with ventricular hypertrophy, and it is therefore possible mutations in the NPPB gene could cause HCM. METHODS: Genomic DNA was extracted from peripheral blood in 238 consecutive probands with HCM. The coding regions and intron/exon boundaries in the NPPB gene were amplified by PCR, and products were screened for sequence variants using high-performance liquid chromatography, followed by direct DNA sequencing. RESULTS: Four sequence variants in the NPPB gene were identified in 9 of the 238 probands screened. Two of the variants were intronic, one was a synonymous variant at codon 79, and the final variant resulted in an amino acid substitution from arginine to histidine at codon 47 (Arg47His). The Arg47His variant was identified in a control population consisting of 204 chromosomes at an allelic frequency of 0.5%, and is therefore unlikely to cause disease. CONCLUSION: No disease causing mutations were identified in the NPPB gene in this cohort, indicating that mutations in this gene are unlikely to be responsible for HCM.     

Rendu-Osler disease: clinical and molecular update

The Rendu-Osler disease, also called Hereditary Hemorrhagic Telangiectasia (HHT) affects 1 in -5-8000 people. A french epidemiological study pointed out that it was particularly high in the Haut-Jura mountains in France. This pathology is characterized by frequent nosebleeds, mucocutaneous and visceral telangiectasia and hereditary autosomal-dominant trait. The mucocutaneous telangiectasia are hemorrhagic while the visceral telangiectasia, less frequent, lead to arteriovenous fistula in the lungs, the liver and the brain. HHT disease-causing genes (ENG, ACVRL1 and MADH4) encode proteins that modulate TGFβ superfamilly signaling in vascular endothelial cells. The recent discovery that BMP9 acts as the specific ligand of the receptor ALK1 and endoglin as its co-receptor shows that this signaling pathway is involved in the maturation phase of angiogenesis. Mice heterozygous for endoglin or ALK1 defects reproduce the HHT phenotype and further support the involvement of endothelial hyper proliferation in the pathogenesis of the disease. The medical management of patients remains mainly symptomatic, however the angiogenic trait of this disease should allow us to consider in the future new -therapeutic approaches using anti-angiogenic drugs.     

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.     

Clustered coding variants in the glutamate receptor complexes of individuals with schizophrenia and bipolar disorder

Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic 'hub' genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.     

How to assess the pathogenicity of mutations in Charcot-Marie-Tooth disease and other diseases?

Knowledge whether a certain DNA variant is a pathogenic mutation or a harmless polymorphism is a critical issue in medical genetics, in which results of a molecular analysis may serve as a basis for diagnosis and genetic counseling. Due to its genetic heterogeneity expressed at the levels of loci, genes and mutations, Charcot-Marie-Tooth (CMT) disease can serve as a model group of clinically homogenous diseases for studying the pathogenicity of mutations. Close to a 17p11.2-p12 duplication occurring in 70% of patients with the demyelinating form of CMT disease, numerous mutations have been identified in poorly characterized genes coding for proteins of an unknown function. Functional analyses, segregation analyses of large pedigrees, and inclusion of large control groups are required to assess the potential pathogenicity of CMT mutations. Hence, the pathogenicity of numerous CMT mutations remains unclear. Some variants detected in the CMT genes and originally described as pathogenic mutations have been shown to have a polymorphic character. In contrast, polymorphisms initially considered harmless were later reclassified as pathogenic mutations. However, the process of assessing the pathogenicity of mutations, as presented in this study for CMT disorders, is a more general issue concerning all disorders with a genetic background. Since the number of DNA variants is still growing, in the near future geneticists will increasingly have to cope with the problem of pathogenicity of identified genetic variants.     

Clinical and molecular findings in osteoporosis-pseudoglioma syndrome

Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.     

Exome Sequencing of 75 Individuals from Multiply Affected Coeliac Families and Large Scale Resequencing Follow Up

Coeliac disease (CeD) is a highly heritable common autoimmune disease involving chronic small intestinal inflammation in response to dietary wheat. The human leukocyte antigen (HLA) region, and 40 newer regions identified by genome wide association studies (GWAS) and dense fine mapping, account for ∼40% of the disease heritability. We hypothesized that in pedigrees with multiple individuals with CeD rare [minor allele frequency (MAF) <0.5%] mutations of larger effect size (odds ratios of ∼ 2–5) might exist. We sequenced the exomes of 75 coeliac individuals of European ancestry from 55 multiply affected families. We selected interesting variants and genes for further follow up using a combination of: an assessment of shared variants between related subjects, a model-free linkage test, and gene burden tests for multiple, potentially causal, variants. We next performed highly multiplexed amplicon resequencing of all RefSeq exons from 24 candidate genes selected on the basis of the exome sequencing data in 2,248 unrelated coeliac cases and 2,230 controls. 1,335 variants with a 99.9% genotyping call rate were observed in 4,478 samples, of which 939 were present in coding regions of 24 genes (Ti/Tv 2.99). 91.7% of coding variants were rare (MAF <0.5%) and 60% were novel. Gene burden tests performed on rare functional variants identified no significant associations (p<1×10⁻³) in the resequenced candidate genes. Our strategy of sequencing multiply affected families with deep follow up of candidate genes has not identified any new CeD risk mutations.     

The Characteristics of Heterozygous Protein Truncating Variants in the Human Genome

Sequencing projects have identified large numbers of rare stop-gain and frameshift variants in the human genome. As most of these are observed in the heterozygous state, they test a gene’s tolerance to haploinsufficiency and dominant loss of function. We analyzed the distribution of truncating variants across 16,260 autosomal protein coding genes in 11,546 individuals. We observed 39,893 truncating variants affecting 12,062 genes, which significantly differed from an expectation of 12,916 genes under a model of neutral de novo mutation (p<10⁻⁴). Extrapolating this to increasing numbers of sequenced individuals, we estimate that 10.8% of human genes do not tolerate heterozygous truncating variants. An additional 10 to 15% of truncated genes may be rescued by incomplete penetrance or compensatory mutations, or because the truncating variants are of limited functional impact. The study of protein truncating variants delineates the essential genome and, more generally, identifies rare heterozygous variants as an unexplored source of diversity of phenotypic traits and diseases.     

Rare variants in APP, PSEN1 and PSEN2 increase risk for AD in late-onset Alzheimer's disease families

Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09×10⁻⁵; OR = 2.21; 95%CI = 1.49–3.28) or an unselected population of 12,481 samples (p = 6.82×10⁻⁵; OR = 2.19; 95%CI = 1.347–3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS.     

Fifteen novel FBN1 mutations causing Marfan syndrome detected by heteroduplex analysis of genomic amplicons.

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause the Marfan syndrome (MFS). This statement is supported by the observations that the classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and that a significant number of FBN1 mutations have been identified in affected individuals. We have now devised a method to screen the entire coding sequence and flanking splice junctions of FBN1. On completion for a panel of nine probands with classic MFS, six new mutations were identified that accounted for disease in seven (78%) of nine patients. Nine additional new mutations have been characterized in the early stages of a larger screening project. These 15 mutations were equally distributed throughout the gene and, with one exception, were specific to single families. One-third of mutations created premature termination codons, and 6 of 15 substituted residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. Mutations causing severe and rapidly progressive disease that presents in the neonatal period can occur in a larger region of the gene than previously demonstrated, and the nature of the mutation is as important a determinant as its location, in predisposing to this phenotype.     

Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome

Birt-Hogg-Dubé syndrome (BHD), a genodermatosis characterized by multiple hamartomas of the hair follicle (fibrofolliculoma), predisposes individuals to an increased risk of developing renal neoplasms and spontaneous pneumothorax. Previously, we localized the BHD locus (also known as FLCN) to chromosome 17p11.2 by linkage analysis and subsequently identified germline mutations in a novel gene in probands from eight of the nine families with BHD in our screening panel. Affected members of five of the families inherited an insertion/deletion of a cytosine in a C8 tract in exon 11. This mutation was also identified by exon 11 screening in probands from 22 of 52 additional families with BHD and therefore represents a hypermutable "hotspot" for mutation in BHD. Here, we screened the remaining 30 families from this large BHD cohort by direct sequence analysis and identified germline BHD mutations in 84% (51/61) of all families with BHD recruited to our study. Mutations were located along the entire length of the coding region, including 16 insertion/deletion, 3 nonsense, and 3 splice-site mutations. The majority of BHD mutations were predicted to truncate the BHD protein, folliculin. Among patients with a mutation in the exon 11 hotspot, significantly fewer renal tumors were observed in patients with the C-deletion than those with the C-insertion mutation. Coding-sequence mutations were not found, however, in probands from two large families with BHD whose affected members shared their family's BHD-affected haplotype. Of the 53 families with BHD whose members inherited either a germline mutation or the affected haplotype, 24 (45%) had at least one member with renal neoplasms. Three families classified with familial renal oncocytoma were identified with BHD mutations, which represents the first disease gene associated with this rare form of renal neoplasm. This study expands the BHD-mutation spectrum and evaluates genotype-phenotype correlations among families with BHD.