The role of the interactome in the maintenance of deleterious variability in human populations

Recent genomic projects have revealed the existence of an unexpectedly large amount of deleterious variability in the human genome. Several hypotheses have been proposed to explain such an apparently high mutational load. However, the mechanisms by which deleterious mutations in some genes cause a pathological effect but are apparently innocuous in other genes remain largely unknown. This study searched for deleterious variants in the 1,000 genomes populations, as well as in a newly sequenced population of 252 healthy Spanish indi-viduals. In addition, variants causative of monogenic diseases and somatic variants from 41 chronic lymphocytic leukaemia patients were analysed. The deleterious variants found were analysed in the context of the interactome to understand the role of network topology in the maintenance of the observe

Deciphering intrafamilial phenotypic variability by exome sequencing in a Bardet–Biedl family

Bardet–Biedl syndrome (BBS) is a model ciliopathy characterized by a wide range of clinical variability. The heterogeneity of this condition is reflected in the number of underlying gene defects and the epistatic interactions between the proteins encoded. BBS is generally inherited in an autosomal recessive trait. However, in some families, mutations across different loci interact to modulate the expressivity of the phenotype. In order to investigate the magnitude of epistasis in one BBS family with remarkable intrafamilial phenotypic variability, we designed an exome sequencing–based approach using SOLID 5500xl platform. This strategy allowed the reliable detection of the primary causal mutations in our family consisting of two novel compound heterozygous mutations in McKusick–Kaufman syndrome (MKKS) gene (p.D90G and p.V396F). Additionally, exome sequencing enabled the detection of one novel heterozygous NPHP4 variant which is predicted to activate a cryptic acceptor splice site and is only present in the most severely affected patient. Here, we provide an exome sequencing analysis of a BBS family and show the potential utility of this tool, in combination with network analysis, to detect disease-causing mutations and second-site modifiers. Our data demonstrate how next-generation sequencing (NGS) can facilitate the dissection of epistatic phenomena, and shed light on the genetic basis of phenotypic variability

Novel RP1 mutations and a recurrent BBS1 variant explain the co-existence of two distinct retinal phenotypes in the same pedigree

Background: Molecular diagnosis of Inherited Retinal Dystrophies (IRD) has long been challenging due to the extensive clinical and genetic heterogeneity present in this group of disorders. Here, we describe the clinical application of an integrated next-generation sequencing approach to determine the underlying genetic defects in a Spanish family with a provisional clinical diagnosis of autosomal recessive Retinitis Pigmentosa (arRP). Results: Exome sequencing of the index patient resulted in the identification of the homozygous BBS1 p.M390R mutation. Sanger sequencing of additional members of the family showed lack of co-segregation of the p.M390R variant in some individuals. Clinical reanalysis indicated co-ocurrence of two different phenotypes in the same family: Bardet-Biedl syndrome in the individual harboring the BBS1 mutation and non-syndromic arRP in extended family members. To identify possible causative mutations underlying arRP, we conducted disease-targeted gene sequencing using a panel of 26 IRD genes. The in-house custom panel was validated using 18 DNA samples known to harbor mutations in relevant genes. All variants were redetected, indicating a high mutation detection rate. This approach allowed the identification of two novel heterozygous null mutations in RP1 (c.4582_4585delATCA; p.I1528Vfs*10 and c.5962dupA; p.I1988Nfs*3) which co-segregated with the disease in arRP patients. Additionally, a mutational screening in 96 patients of our cohort with genetically unresolved IRD revealed the presence of the c.5962dupA mutation in one unrelated family. Conclusions: The combination of molecular findings for RP1 and BBS1 genes through exome and gene panel sequencing enabled us to explain the co-existence of two different retinal phenotypes in a family. The identification of two novel variants in RP1 suggests that the use of panels containing the prevalent genes of a particular population, together with an optimized data analysis pipeline, is an efficient and cost-effective approach that can be reliably implemented into the routine diagnostic process of diverse inherited retinal disorders. Moreover, the identification of these novel variants in two unrelated families supports the relatively high prevalence of RP1 mutations in Spanish population and the role of private mutations for commonly mutated genes, while extending the mutational spectrum of RP1Instituto de Salud Carlos III (ISCIII)Spanish Ministry of Economy and Competitiveness, Spain (PI1102923, CIBERER ACCI and CDTI FEDER-Innterconecta EXP00052887/ITC-20111037)Foundation Ramón Areces (CIVP16A1856)ISCIII fellowship FI12/0054

Deciphering intrafamilial phenotypic variability by exome sequencing in a Bardet–Biedl family

Bardet–Biedl syndrome (BBS) is a model ciliopathy characterized by a wide range of clinical variability. The heterogeneity of this condition is reflected in the number of underlying gene defects and the epistatic interactions between the proteins encoded. BBS is generally inherited in an autosomal recessive trait. However, in some families, mutations across different loci interact to modulate the expressivity of the phenotype. In order to investigate the magnitude of epistasis in one BBS family with remarkable intrafamilial phenotypic variability, we designed an exome sequencing–based approach using SOLID 5500xl platform. This strategy allowed the reliable detection of the primary causal mutations in our family consisting of two novel compound heterozygous mutations in McKusick–Kaufman syndrome (MKKS) gene (p.D90G and p.V396F). Additionally, exome sequencing enabled the detection of one novel heterozygous NPHP4 variant which is predicted to activate a cryptic acceptor splice site and is only present in the most severely affected patient. Here, we provide an exome sequencing analysis of a BBS family and show the potential utility of this tool, in combination with network analysis, to detect disease-causing mutations and second-site modifiers. Our data demonstrate how next-generation sequencing (NGS) can facilitate the dissection of epistatic phenomena, and shed light on the genetic basis of phenotypic variabilityInstituto de Salud Carlos III (ISCIII)Spanish Ministry of Economy and Competitiveness (PI1102923)Regional Ministry of Economy, Innovation, Science and Employment of the Autonomous Government of Andalusia (CTS-03687)Regional Ministry of Health of the Autonomous Government of Andalusia (PI100154), (PCT-30000-2009-12)INNPLANTA (PCT-300000-2010-007)Ciber de Enfermedades raras (CIBERER)Foundation Ramon Areces (CIVP16A1856)Spanish Ministry of Science and Innovation (BIO2011-27069)Conselleria de Educacio of the Valencia Community (PROMETEO/2010/001

Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

<div><p>This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in <i>RHO</i>, c.937-2_944del, (ii) one rare homozygous mutation in <i>C2orf71</i>, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in <i>ABCA4,</i> c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of <i>RPGR</i>. The molecular findings for <i>RHO</i> and <i>C2orf71</i> confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two <i>ABCA4</i> mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the <i>RPGR</i> mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.</p></div

Clinical characteristics of affected family members.

<p># Consanguineous family</p>†<p>Previously reported by Maugeri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Maugeri1" target="_blank">[24]</a> in a Stargardt patient in heterozygous state.</p>‡<p>Previously reported by Lewis et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Lewis1" target="_blank">[25]</a> in a Stargardt patient in heterozygous state.</p>¥<p>Previously reported by Vervoort et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116176#pone.0116176-Vervoort1" target="_blank">[23]</a>.</p><p>N.A.: Not available.</p><p>N.R.: Not recordable.</p><p>Clinical characteristics of affected family members.</p

Segregation studies of identified variants in the four analyzed families.

<p>Below the individuals, genotypes are presented for each change segregating with RP. Index patients are indicated with a black arrow. [M];[M] represents homozygous mutants; [M];[ = ] indicates heterozygous carriers, [ = ];[ = ] indicates individuals carrying two wild-type alleles, whereas [m] represents hemizygous individuals. NA means non available DNA sample. W means samples processed by WES.</p

Novel pathogenic variants identified in this study.

<p>A) Chromatograms of wild type (WT) <i>RHO</i> DNA sequence (NM_000539.3) and RP subject (MUT) showing the heterozygous mutation c.937-2_944delAGTTCCGGAA. SAS: Splice Acceptor Site. Exon 5 is highlighted in blue. B) The <i>RHO</i> gene structure is composed of 5 exons that are indicated as filled boxes while 5′ and 3′ UTRs are shown as open boxes. The canonical SAS of exon 5 is in blue and the predicted cryptic SAS (CSAS) is in red and indicated with an asterisk. The WT protein product (left) has 348 aminoacids (aa) while the predicted mutant product (MUT) only 326 aa. C) Chromatograms of WT <i>C2orf71</i> (NM_001029883.2) DNA sequence and RP subject (MUT) showing the homozygous mutation c.1795T>C. D) Alignment of C2orf71 orthologous protein sequences showing conservation of the mutated residue p.Cys599Arg among mammals. E) Prediction of disulfide bonds formation for WT and mutant (MUT) C2orf71 proteins. According to the prediction, in the mutant protein structure, two of the disulfide bonds have been disrupted while a new one has been created. The disulfide bonds altered are in red.</p

267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation.

Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms