Candidate Genes In Families Research Articles

Research Techniques Made Simple: Whole-Transcriptome Sequencing by RNA-Seq for Diagnosis of Monogenic Disorders

Mendelian disorders with cutaneous manifestations comprise a genotypically heterogeneous group of over 1,000 diseases, and in most of them mutant genes have been identified. Mutation detection approaches in these diseases have largely focused on DNA analysis by next-generation sequencing techniques, including gene-targeted sequencing panels as well as whole-exome and whole-genome sequencing. Genome-wide homozygosity mapping (HM), based on DNA polymorphism, has also assisted in the identification of candidate genes in families with consanguinity. However, specific pathogenic variants have not been disclosed in many individual patients when analyzed by next-generation sequencing, and in particular, DNA-based analysis failed to identify many of the mutations impacting on splicing or gene expression. Whole-transcriptome sequencing by RNA sequencing (RNA-Seq), with appropriate bioinformatics, provides a robust tool to identify additional mutations to facilitate genetic diagnosis in genodermatoses. RNA-Seq can be used for variant calling and HM similar to DNA-based approaches, but it also allows for the identification of mutations that result in aberrant transcriptome expression, as displayed by heatmap analysis, and altered splicing patterns of RNA, as visualized by Sashimi plots. Thus, clinical RNA-Seq extends molecular diagnostics of rare genodermatoses, and it could provide a reliable first-tier diagnostic approach to extend mutation databases in patients with heritable skin diseases.

A comprehensive analysis of candidate genes in familial pancreatic cancer families reveals a high frequency of potentially pathogenic germline variants

BackgroundThe 5-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is around 5% due to the fact that the majority of patients present with advanced disease that is treatment resistant. Familial pancreatic cancer (FPC) is a rare disorder that is defined as a family with at least two affected first degree relatives, with an estimated incidence of 4%–10%. The genetic basis is unknown in the majority of families although around 10%–13% of families carry germline mutations in known genes associated with hereditary cancer and pancreatitis syndromes.MethodsPanel sequencing was performed of 35 genes associated with hereditary cancer in 43 PDAC cases from families with an apparent hereditary pancreatic cancer syndrome. Findings: Pathogenic variants were identified in 19% (5/26) of PDAC cases from pure FPC families in the genes MLH1, CDKN2A, POLQ and FANCM. Low frequency potentially pathogenic VUS were also identified in 35% (9/26) of PDAC cases from FPC families in the genes FANCC, MLH1, PMS2, CFTR, APC and MUTYH. Furthermore, an important proportion of PDAC cases harboured more than one pathogenic, likely pathogenic or potentially pathogenic VUS, highlighting the multigene phenotype of FPC.InterpretationThe genetic basis of familial or hereditary pancreatic cancer can be explained in 21% of families by previously described hereditary cancer genes. Low frequency variants in other DNA repair genes are also present in 35% of families which may contribute to the risk of pancreatic cancer development.FundingThis study was funded by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013–2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund ‘‘A way to achieve Europe’’ (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Temática de investigación cooperativa en cáncer: RTICC (RD12/0036/0073) and La Asociación Española contra el Cáncer: AECC (Grupos Coordinados Estables 2016).

Genetic testing of Mucopolysaccharidoses disease using multiplex PCR- based panels of STR markers: in silico analysis of novel mutations.

The Mucopolysaccharidoses (MPS) are group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade glycosaminoglycans (GAGs) in the lysosomes. GAGs are sulfated polysaccharides involving repeating disaccharides, uronic acid and hexosamines including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). Hyaluronan is excluded in terms of being non-sulfated in the GAG family. Different types of mutations have been identified as the causative agent in all types of MPS. Herein, we planned to investigate the pathogenic mutations in different types of MPS including type I (IDUA gene), IIIA (SGSH) and IIIB (NAGLU) in the eight Iranian patients. Autozygosity mapping was performed to identify the potential pathogenic variants in these 8 patients indirectly with the clinical diagnosis of MPSs. so three panels of STR (Short Tandem Repeat) markres flanking IDUA, SGSH and NAGLU genes were selected for multiplex PCR amplification. Then in each family candidate gene was sequenced to identify the pathogenic mutation. Our study showed two novel mutations c.469T > C and c.903C > G in the IDUA gene, four recurrent mutations: c.1A > C in IDUA, c.220C > T, c.1298G > A in SGSH gene and c.457G > A in the NAGLU gene. The c.1A > C in IDUA was the most common mutation in our study. In silico analysis were performed as well to predict the pathogenicity of the novel variants.

Exploring digenic inheritance in arrhythmogenic cardiomyopathy.

BackgroundArrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder, characterized by the substitution of heart muscle with fibro-fatty tissue and severe ventricular arrhythmias, often leading to heart failure and sudden cardiac death. ACM is considered a monogenic disorder, but the low penetrance of mutations identified in patients suggests the involvement of additional genetic or environmental factors.MethodsWe used whole exome sequencing to investigate digenic inheritance in two ACM families where previous diagnostic tests have revealed a PKP2 mutation in all affected and some healthy individuals. In family members with PKP2 mutations we determined all genes that harbor variants in affected but not in healthy carriers or vice versa. We computationally prioritized the most likely candidates, focusing on known ACM genes and genes related to PKP2 through protein interactions, functional relationships, or shared biological processes.ResultsWe identified four candidate genes in family 1, namely DAG1, DAB2IP, CTBP2 and TCF25, and eleven candidate genes in family 2. The most promising gene in the second family is TTN, a gene previously associated with ACM, in which the affected individual harbors two rare deleterious-predicted missense variants, one of which is located in the protein’s only serine kinase domain.ConclusionsIn this study we report genes that might act as digenic players in ACM pathogenesis, on the basis of co-segregation with PKP2 mutations. Validation in larger cohorts is still required to prove the utility of this model.

POLR2C Mutations Are Associated With Primary Ovarian Insufficiency in Women.

Context:Primary ovarian insufficiency (POI) results from a premature loss of oocytes, causing infertility and early menopause. The etiology of POI remains unknown in a majority of cases.Objective:To identify candidate genes in families affected by POI.Design:This was a family-based genetic study.Setting:The study was performed at two academic institutions.Patients and Other Participants:A family with four generations of women affected by POI (n = 5). Four of these women, three with an associated autoimmune diagnosis, were studied. The controls (n = 387) were recruited for health in old age.Intervention:Whole-genome sequencing was performed.Main Outcome Measure:Candidate genes were identified by comparing gene mutations in three family members and 387 control subjects analyzed simultaneously using the pedigree Variant Annotation, Analysis and Search Tool. Data were also compared with that in publicly available databases.Results:We identified a heterozygous nonsense mutation in a subunit of RNA polymerase II (POLR2C) that synthesizes messenger RNA. A rare sequence variant in POLR2C was also identified in one of 96 women with sporadic POI. POLR2C expression was decreased in the proband compared with women with POI from another cause. Knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation.Conclusions:These data support a role for RNA polymerase II mutations as candidates in the etiology of POI. The current data also support results from genome-wide association studies that hypothesize a role for RNA polymerase II subunits in age at menopause in the population.

RARE VARIANTS IN THE CYP27B1 GENE ARE ASSOCIATED WITH MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is a complex neurological disease. Genetic linkage analysis and genotyping of candidate genes in families with four or more affected individuals more heavily loaded for susceptibility genes...

A missense mutation in CRYGD linked with autosomal dominant congenital cataract of aculeiform type

To detect the underlying genetic defects in two autosomal dominant congenital cataract (ADCC) families, having respectively twenty and four members affected with bilateral congenital cataract. Detailed family history and clinical data were recorded. Mutation screening in twenty three candidate genes including crystallins (CRYAA, CRYAB, CRYBA1/A3, CRYBA2, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGA, CRYGB, CRYGC, CRYGD, and CRYGS), gap junctional channels; connexins (GJA8, GJA3), beaded filament chain proteins (BFSP1, BFSP2), major intrinsic protein (MIP), lens intrinsic membrane protein-2 (LIM2), transcriptional factor (MAF), and in genes encoding for membrane-associated proteins (TMEM114, CHMP4B, EPHA2) was performed by bi-directional sequence analysis of the amplified products. In family A twenty members in six generations were affected by bilateral aculeiform type cataract and in family B four affected members in three generations had granular nuclear cataract. Mutation screening in already known candidate genes by sequence analyses revealed proline to threonine substitution at codon 23 (p.Pro23Thr) in CRYGD for aculeiform type cataract in family A. The family B with four members affected by granular nuclear cataract, however, could not be linked with any of these analyzed 23 candidate genes. The present study describes identification of p.Pro23Thr mutation in CRYGD for aculeiform type cataract in an ADCC family of Indian origin. The identical mutation has previously been reported to be linked with different phenotypes; lamellar cataract, cerulean cataract, coralliform cataract, flaky silica-like nuclear cataract and fasciculiform type cataract in different ADCC families. Interestingly, a mutation of different codon, i.e., p.Arg58His in CRYGD has been reported to be linked with aculeiform cataract in four different families; two from Switzerland, one from Macedonia and in a Mexican family. The findings in present study thus expand the genetic heterogeneity for aculeiform type cataract. Further, exclusion of these twenty three known candidate genes in family B having ADCC of granular nuclear type indicates the role of some other gene apart from for crystallins, gap junction channels, beaded filaments and membrane-associated proteins, and MAF for this phenotype.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Rare variants in the CYP27B1 gene are associated with multiple sclerosis

Multiple sclerosis (MS) is a complex neurological disease. Genetic linkage analysis and genotyping of candidate genes in families with 4 or more affected individuals more heavily loaded for susceptibility genes has not fully explained familial disease clustering. We performed whole exome sequencing to further understand the heightened prevalence of MS in these families. Forty-three individuals with MS (1 from each family) were sequenced to find rare variants in candidate MS susceptibility genes. On average, >58,000 variants were identified in each individual. A rare variant in the CYP27B1 gene causing complete loss of gene function was identified in 1 individual. Homozygosity for this mutation results in vitamin D-dependent rickets I (VDDR1), whereas heterozygosity results in lower calcitriol levels. This variant showed significant heterozygous association in 3,046 parent-affected child trios (p = 1 × 10(-5)). Further genotyping in >12,500 individuals showed that other rare loss of function CYP27B1 variants also conferred significant risk of MS, Peto odds ratio = 4.7 (95% confidence interval, 2.3-9.4; p = 5 × 10(-7)). Four known VDDR1 mutations were identified, all overtransmitted. Heterozygous parents transmitted these alleles to MS offspring 35 of 35× (p = 3 × 10(-9)). A causative role for CYP27B1 in MS is supported; the mutations identified are known to alter function having been shown in vivo to result in rickets when 2 copies are present. CYP27B1 encodes the vitamin D-activating 1-alpha hydroxylase enzyme, and thus a role for vitamin D in MS pathogenesis is strongly implicated.

Screening of functional and positional candidate genes in families with common variable immunodeficiency

BackgroundCommon variable immunodeficiency (CVID) comprises a heterogeneous group of primary antibody deficiencies with complex clinical and immunological phenotypes. The recent discovery that some CVID patients show monogenic defects in the genes encoding ICOS, TACI or CD19 prompted us to investigate several functional candidate genes in individuals with CVID.ResultsThe exonic, protein coding regions of the genes encoding: APRIL, BCMA, IL10, IL10Rα, IL10Rβ, IL21, IL21R, and CCL18, were analyzed primarily in familial CVID cases, who showed evidence of genetic linkage to the respective candidate gene loci and CVID families with a recessive pattern of inheritance. Two novel SNPs were identified in exon 5 and exon 8 of the IL21R gene, which segregated with the disease phenotype in one CVID family. Eleven additional SNPs in the genes encoding BCMA, APRIL, IL10, IL10Rα, IL21 and IL21R were observed at similar frequencies as in healthy donors.ConclusionWe were unable to identify obvious disease causing mutations in the protein coding regions of the analyzed genes in the studied cohort.

Genetic linkage of candidate genes in families with abdominal aortic aneurysms?

Objectives: to examine possible involvement of several candidate genes in the aetiology of familial abdominal aortic aneurysm (AAA). Design: after reviewing the literature on the genetics of familial AAA, betaine homocysteine methyltransferase (BHMT), collagen type Iα2 (COL1A2) and cathepsin H (CTSH), were selected as potential candidate genes, which influence structure, strength, elasticity and mechanical resistance of the aortic wall. Materials: forty-eight families with 110 family members and AAA were included in the affected sib-pair analysis. One large family of three generations was analysed separately because in this family also other clinical symptoms were involved. Methods: genetic linkage analysis was performed with DNA markers in the region of BHMT, COL1A2 and CTSH. Results: In the overall sib-pair analysis, the LOD scores for BHMT, COL1A2 and CTSH were 0.7, 0.2 and −0.7, whereas in the large family these numbers were −0.6, −2.2 and −2.7, respectively. Conclusions: none of the candidate genes selected showed a suggestive linkage with AAA. Exclusion of the COL1A2 and CTSH genes was possible in the large family that was analysed separately.Eur J Vasc Endovasc Surg 26, 205-210 (2003)

Design considerations for association studies of candidate genes in families.

In genetic epidemiologic studies, investigators often use generalized linear models to evaluate the relationships between a disease trait and covariates, such as one or more candidate genes or an environmental exposure. Recently, attention has turned to study designs that mandate the inclusion of family members in addition to a proband. Standard models for analysis assume independent observations, which is unlikely to be true for family data, and the usual standard errors for the regression parameter estimates may be too large or too small, depending on the distribution of the covariates within and between families. The consequences of familial correlation on the study efficiency can be measured by a design effect that is equivalent to the relative information in a sample of unrelated individuals compared to a sample of families with the same number of individuals. We examine design effects for studies in association, and illustrate how the design effect is influenced by the intra-familial distribution of covariate values such as would be expected for a candidate gene. Typical design effects for a candidate gene range between 1.1 and 2.4, depending on the size of the family and the amount of unexplained familial correlation. These values correspond to a modest 10% increase in the required sample size up to more than doubling the requirements. Design effect values are useful in study design to compare the efficiency of studies that sample families versus independent individuals and to determine sample size requirements that account for familial correlation.

Genetics of inflammatory bowel disease: implications for the future.

There is overwhelming evidence that genetic factors play a role in the predisposition to suffer the development of the chronic inflammatory bowel diseases. The genetic analysis of complex diseases, such as ulcerative colitis and Crohn's disease, is difficult. The presence of disease heterogeneity, the relative low frequency in the population, the degree to which first-degree relatives are affected (approximately 10%), the presence of genes with minor genetic effects, and ethnic differences are some of the difficulties encountered when identifying disease susceptibility loci. Two major approaches to identifying these genes are being followed at present. The first, family-based, consists of studying linkage analysis in sibling pairs and parental transmission in genome-wide screening using microsatellite markers. These studies are appropriate and helpful for finding genes of major or moderate effects but may provide difficulty when identifying genes with minor effects. Risch and Merikangas have pointed to the power of association studies utilizing candidate genes in families. These studies should be considered in the future in genome-wide screens when technologic advances permit. The second approach is based on classic epidemiologic designs, population-based studies, using candidate genes in the framework of a biologic hypothesis. Recent data using both approaches in both Crohn's disease and ulcerative colitis are reviewed. The results of genome-wide linkage studies have not reached consensus but suggest that these diseases are different and polygenic in nature. We have started our studies with the hypothesis that an abnormal immune dysbalance contributes to the biologic basis of disease. We therefore study polymorphisms in genes encoding proinflammatory and regulatory cytokines. Preliminary data of these association studies suggest the importance of several genes with small effects in determining the severity and prognosis of these diseases.