Targeted mutation screening of 292 candidate genes in 38 children with inborn haematological cytopenias efficiently identifies novel disease-causing mutations.

Br J Haematol. 2018 May 24;:

Authors: Kager L, Jimenez Heredia R, Hirschmugl T, Dmytrus J, Krolo A, Müller H, Bock C, Zeitlhofer P, Dworzak M, Mann G, Holter W, Haas O, Boztug K

Abstract
Establishing a precise diagnosis is essential in inborn haematological cytopenias to enable appropriate treatment decisions and avoid secondary organ damage. However, both diversity and phenotypic overlap of distinct disease entities may make the identification of underlying genetic aetiologies by classical Sanger sequencing challenging. Instead of exome sequencing, we established a systematic next generation sequencing-based panel targeting 292 candidate genes and screened 38 consecutive patients for disease-associated mutations. Efficient identification of the underlying genetic cause in 17 patients (44·7%), including 13 novel mutations, demonstrates that this approach is time- and cost-efficient, enabling optimal management and genetic counselling.

PMID: 29797310 [PubMed - as supplied by publisher]

Insights into the genetic epidemiology of Crohn's and rare diseases in the Ashkenazi Jewish population.

PLoS Genet. 2018 May;14(5):e1007329

Authors: Rivas MA, Avila BE, Koskela J, Huang H, Stevens C, Pirinen M, Haritunians T, Neale BM, Kurki M, Ganna A, Graham D, Glaser B, Peter I, Atzmon G, Barzilai N, Levine AP, Schiff E, Pontikos N, Weisburd B, Lek M, Karczewski KJ, Bloom J, Minikel EV, Petersen BS, Beaugerie L, Seksik P, Cosnes J, Schreiber S, Bokemeyer B, Bethge J, International IBD Genetics Consortium, NIDDK IBD Genetics Consortium, T2D-GENES Consortium, Heap G, Ahmad T, Plagnol V, Segal AW, Targan S, Turner D, Saavalainen P, Farkkila M, Kontula K, Palotie A, Brant SR, Duerr RH, Silverberg MS, Rioux JD, Weersma RK, Franke A, Jostins L, Anderson CA, Barrett JC, MacArthur DG, Jalas C, Sokol H, Xavier RJ, Pulver A, Cho JH, McGovern DPB, Daly MJ

Abstract
As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10-100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p<10-16). Taken together, the results suggest coordinated selection in AJ population for higher CD risk alleles in general. The results and approach illustrate the value of exome sequencing data in case-control studies along with reference data sets like ExAC (sites VCF available via FTP at ftp.broadinstitute.org/pub/ExAC_release/release0.3/) to pinpoint genetic variation that contributes to variable disease predisposition across populations.

PMID: 29795570 [PubMed - in process]

Identification of HLA-DQA1 as a Susceptibility Gene for Spinal Tuberculosis by Exome Sequencing.

Med Sci Monit. 2018 May 24;24:3442-3449

Authors: Shen J, Shi S, Lai Z

Abstract
BACKGROUND Spinal tuberculosis (STB) is the main cause of bone and joint tuberculosis. This study aimed to screen and analyze the susceptibility genes for STB using whole-exome sequencing (WES). MATERIAL AND METHODS All exon regions of peripheral blood DNA from 6 STB patients were captured and sequenced using WES and the sequencing data were analyzed by modern bioinformatics methods to identify disease-causing mutations. Sanger sequencing was then used to validate the mutation sites in normal controls (207) and STB patients (193). The mRNA expression of the mutant gene and the serum levels of IL-6 and TNF-α were detected using qPCR or ELISA assay, respectively. RESULTS A nonsynonymous single-nucleotide polymorphism (SNP) in the gene HLA-DQA1 (rs796778515, c.592delCinsG, CAG to GAG, p.Q198E) was identified and further validated by Sanger sequencing. The percentage of the 3 genotypes C/C, C/G and G/G in STB patients and normal controls were 37.3%, 32.1%, and 30.6% and 47.8%, 33.8%, and 18.4%, respectively. Furthermore, the C>G mutation was significantly associated with the occurrence of STB. In addition, the levels of HLA-DQA1 mRNA were significantly lower in blood cells from STB patients compared with normal controls, while the serum levels of IL-6 and TNF-α were significantly higher. CONCLUSIONS The C>G mutation in the HLA-DQA1 gene was associated with the occurrence of STB. This variation may result in the decreased level of HLA-DQA1 mRNA and increased serum levels of IL-6 and TNF-α, which finally led the STB susceptibility.

PMID: 29795056 [PubMed - in process]

Tiled array-based sequencing identifies enrichment of loss-of-function variants in the highly homologous filaggrin gene in African American children with severe atopic dermatitis.

Exp Dermatol. 2018 May 23;:

Authors: Mathyer ME, Quiggle AM, Wong XFCC, Denil SLIJ, Kumar MG, Ciliberto HM, Bayliss SJ, Common JE, de Guzman Strong C

Abstract
Filaggrin (FLG) loss of function (LOF) variants are a major risk factor for the common inflammatory skin disease, atopic dermatitis (AD), and are often population-specific. African American (AA) children are disproportionately affected with AD, often later developing asthma and/or allergic rhinitis and comprise an atopy health disparity group for which the role of FLG LOF is not well known. Discovery of FLG LOF using exome sequencing is challenging given the known difficulties for accurate short-read alignment to FLG's high homology repeat variation. Here we employed an array-based sequencing approach to tile across each FLG repeat and discover FLG LOF in a well-characterized cohort of AA children with moderate to severe AD. Five FLG LOF were identified in 23% of our cohort. Two novel FLG LOF singletons, c.488delG and p.S3101*, were discovered as well as p.R501*, p.R826*, and p.S3316* previously reported for AD. p.S3316* (rs149484917) is likely an African ancestral FLG LOF, reported in African individuals in ExAC (Exome Aggregation Consortium), Exome Variant Server (ESP), and 4 African 1000G population databases (ESN, MSL, ASW, and ACB). The proportion of FLG LOF (11.5%) among the total FLG alleles in our cohort was significantly higher in comparisons to FLG LOF reported for African individuals in ExAC (2.5%; P = 4.3x10-4 ) and ESP (1.7%; P = 3.5x10-5 ) suggesting a disease-enrichment effect for FLG LOF. Our results demonstrate the utility of array-based sequencing in discovering FLG LOF, including novel and population-specific, that are of higher prevalence in our AA severe AD group than previously reported. This article is protected by copyright. All rights reserved.

PMID: 29791750 [PubMed - as supplied by publisher]

Point-of-care whole-exome sequencing of idiopathic male infertility.

Genet Med. 2018 Apr 12;:

Authors: Fakhro KA, Elbardisi H, Arafa M, Robay A, Rodriguez-Flores JL, Al-Shakaki A, Syed N, Mezey JG, Abi Khalil C, Malek JA, Al-Ansari A, Al Said S, Crystal RG

Abstract
PurposeNonobstructive azoospermia (NOA) affects 1% of the male population; however, despite state-of-the-art clinical assessment, for most patients the cause is unknown. We capitalized on an analysis of multiplex families in the Middle East to identify highly penetrant genetic causes.MethodsWe used whole-exome sequencing (WES) in 8 consanguineous families and combined newly discovered genes with previously reported ones to create a NOA gene panel, which was used to identify additional variants in 75 unrelated idiopathic NOA subjects and 74 fertile controls.ResultsIn five of eight families, we identified rare deleterious recessive variants in CCDC155, NANOS2, SPO11, TEX14, and WNK3 segregating with disease. These genes, which are novel to human NOA, have remarkable testis-specific expression, and murine functional evidence supports roles for them in spermatogenesis. Among 75 unrelated NOA subjects, we identified 4 (~5.3%) with additional recessive variants in these newly discovered genes and 6 with deleterious variants in previously reported NOA genes, yielding an overall genetic etiology for 13.3% subjects versus 0 fertile controls (p = 0.001).ConclusionNOA affects millions of men, many of whom remain idiopathic despite extensive laboratory evaluation. The genetic etiology for a substantial fraction of these patients (>50% familial and >10% sporadic) may be discovered by WES at the point of care.Genetics in Medicine advance online publication, 12 April 2018; doi:10.1038/gim.2018.10.

PMID: 29790874 [PubMed - as supplied by publisher]

Phenotypic expansion illuminates multilocus pathogenic variation.

Genet Med. 2018 Apr 26;:

Authors: Karaca E, Posey JE, Coban Akdemir Z, Pehlivan D, Harel T, Jhangiani SN, Bayram Y, Song X, Bahrambeigi V, Yuregir OO, Bozdogan S, Yesil G, Isikay S, Muzny D, Gibbs RA, Lupski JR

Abstract
PurposeMultilocus variation-pathogenic variants in two or more disease genes-can potentially explain the underlying genetic basis for apparent phenotypic expansion in cases for which the observed clinical features extend beyond those reported in association with a "known" disease gene.MethodsAnalyses focused on 106 patients, 19 for whom apparent phenotypic expansion was previously attributed to variation at known disease genes. We performed a retrospective computational reanalysis of whole-exome sequencing data using stringent Variant Call File filtering criteria to determine whether molecular diagnoses involving additional disease loci might explain the observed expanded phenotypes.ResultsMultilocus variation was identified in 31.6% (6/19) of families with phenotypic expansion and 2.3% (2/87) without phenotypic expansion. Intrafamilial clinical variability within two families was explained by multilocus variation identified in the more severely affected sibling.ConclusionOur findings underscore the role of multiple rare variants at different loci in the etiology of genetically and clinically heterogeneous cohorts. Intrafamilial phenotypic and genotypic variability allowed a dissection of genotype-phenotype relationships in two families. Our data emphasize the critical role of the clinician in diagnostic genomic analyses and demonstrate that apparent phenotypic expansion may represent blended phenotypes resulting from pathogenic variation at more than one locus.Genetics in Medicine advance online publication, 26 April 2018; doi:10.1038/gim.2018.33.

PMID: 29790871 [PubMed - as supplied by publisher]

Genetics of non-syndromic childhood obesity and the use of high-throughput DNA sequencing technologies.

J Diabetes Complications. 2017 Oct;31(10):1549-1561

Authors: da Fonseca ACP, Mastronardi C, Johar A, Arcos-Burgos M, Paz-Filho G

Abstract
BACKGROUND: Childhood obesity is a serious public health problem associated with the development of several chronic diseases, such as type 2 diabetes mellitus, dyslipidemia, and hypertension. The elevated prevalence of obesity is mostly due to inadequate diet and lifestyle, but it is also influenced by genetic factors.
OBJECTIVES: To review recent advances in the field of the genetics of obesity. We summarize the list of genes associated with the rare non-syndromic forms of obesity, and explain their function. Furthermore, we discuss the technologies that are available for the genetic diagnosis of obesity.
RESULTS: Several studies reported that single gene variants cause Mendelian forms of obesity, determined by mutations of major effect in single genes. Rare, non-syndromic forms of obesity are a result of loss-of-function mutations in genes that act on the development and function of the hypothalamus or the leptin-melanocortin pathway. These variants disrupt enzymes and receptors that play a role in energy homeostasis, resulting in severe early-onset obesity and endocrine dysfunctions. Different approaches and technologies have been used to understand the genetic background of obesity. Currently, whole genome and whole exome sequencing are important diagnostic tools to identify new genes and variants associated with severe obesity, but other approaches are also useful at individual or population levels, such as linkage analysis, candidate gene sequencing, chromosomal microarray analysis, and genome-wide association studies.
CONCLUSIONS: The understanding of the genetic causes of obesity and the usefulness and limitations of the genetic diagnostic approaches can contribute to the development of new personalized therapeutic targets against obesity.

PMID: 28735903 [PubMed - indexed for MEDLINE]

Molecular Screening for Cancer Treatment Optimization (MOSCATO-01) in Pediatric Patients: A Single-Institutional Prospective Molecular Stratification Trial.

Clin Cancer Res. 2017 Oct 15;23(20):6101-6112

Authors: Harttrampf AC, Lacroix L, Deloger M, Deschamps F, Puget S, Auger N, Vielh P, Varlet P, Balogh Z, Abbou S, Allorant A, Valteau-Couanet D, Sarnacki S, Gamiche-Rolland L, Meurice G, Minard-Colin V, Grill J, Brugieres L, Dufour C, Gaspar N, Michiels S, Vassal G, Soria JC, Geoerger B

Abstract
Purpose: This single-institutional feasibility study prospectively characterized genomic alterations in recurrent or refractory solid tumors of pediatric patients to select a targeted therapy.Experimental Design: Following treatment failure, patients with signed consent and ages above 6 months, underwent tumor biopsy or surgical resection of primary or metastatic tumor site. These newly acquired samples were analyzed by comparative genomic hybridization array, next-generation sequencing for 75 target genes, whole-exome and RNA sequencing. Biological significance of the alterations and suggestion of most relevant targeted therapies available were discussed in a multidisciplinary tumor board.Results: From December 2012 to January 2016, 75 patients were included, 73 patients underwent 79 interventions, 56 of which were research biopsies with a low complication rate. All patients were pretreated, 37.0% had a brain tumor, and 63.0% had an extra-cranial solid tumor. Median tumor cell content was 70% (range, 0%-100%). Successful molecular analysis in 69 patients detected in 60.9% of patients an actionable alteration in various oncogenic pathways (42.4% with copy-number change, 33.3% with mutation, 2.1% with fusion), and change in diagnosis in three patients. Fourteen patients received 17 targeted therapies; two had received a matched treatment before inclusion.Conclusions: Research biopsies are feasible in advanced pediatric malignancies that exhibit a considerable amount of potentially actionable alterations. Genetic events affecting different cancer hallmarks and limited access to targeted agents within pediatric clinical trials remain the main obstacles that are addressed in our two subsequent precision medicine studies MAPPYACTS and AcSé-ESMART. Clin Cancer Res; 23(20); 6101-12. ©2017 AACR.

PMID: 28733441 [PubMed - indexed for MEDLINE]

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Clin Cancer Res. 2017 Oct 15;23(20):6315-6324

Authors: Remacha L, Comino-Méndez I, Richter S, Contreras L, Currás-Freixes M, Pita G, Letón R, Galarreta A, Torres-Pérez R, Honrado E, Jiménez S, Maestre L, Moran S, Esteller M, Satrústegui J, Eisenhofer G, Robledo M, Cascón A

Abstract
Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.

PMID: 28720665 [PubMed - indexed for MEDLINE]

Umbilical Cord Blood Transplantation Corrects Very Early-Onset Inflammatory Bowel Disease in Chinese Patients With IL10RA-Associated Immune Deficiency.

Inflamm Bowel Dis. 2018 May 18;:

Authors: Peng K, Qian X, Huang Z, Lu J, Wang Y, Zhou Y, Wang H, Wu B, Wang Y, Chen L, Zhai X, Huang Y

Abstract
Background: Hematopoietic stem cell transplantation is considered the only curative therapy for very early-onset inflammatory bowel disease with specific immune defects, such as interleukin-10 receptor deficiency. We performed reduced-intensity conditioning before umbilical cord blood transplantation in patients with interleukin-10 receptor-A deficiency.
Methods: We enrolled 9 very early-onset inflammatory bowel disease patients with typical manifestations. We diagnosed the patients with interleukin-10 receptor-A deficiency by whole-exome sequencing. Umbilical cord blood transplantation was performed in all 9 patients. Eight patients received the reduced-intensity conditioning regimen, and 1 patient received the myeloablative conditioning regimen.
Results: All 9 patients received transplantation between the ages of 6 months to 43 months (average, 16.8 months) with body weights ranging from 3 to 10.4 kg (average, 6.6 kg). The patients displayed complete chimerism at 2-8 weeks after transplantation; 6 patients achieved complete remission without evidence of graft-vs-host disease or infections; 1 patient died of chronic lung graft-vs-host disease at 6 months post-transplantation; and the other 2 patients died of sepsis post-transplantation because of unsuccessful engraftments. Severe malnutrition and growth retardation associated with interleukin-10 receptor-A deficiency were significantly improved post-transplantation.
Conclusions: We recommend umbilical cord blood transplantation as a potential treatment for very early-onset inflammatory bowel disease with a defined monogenic immunodeficiency, and we suggest that reduced-intensity conditioning chemotherapy is more suitable than myeloablative conditioning for patients with severe malnutrition and bowel disease. We have demonstrated success with reduced-intensity conditioning for interleukin-10 receptor-A deficiency in pediatric patients with severe clinical conditions. 10.1093/ibd/izy028_video1izy028.video15786489183001.

PMID: 29788474 [PubMed - as supplied by publisher]

Infantile Onset Intractable Inflammatory Bowel Disease Due to Novel Heterozygous Mutations in TNFAIP3 (A20).

Inflamm Bowel Dis. 2018 May 17;:

Authors: Zheng C, Huang Y, Ye Z, Wang Y, Tang Z, Lu J, Wu J, Zhou Y, Wang L, Huang Z, Yang H, Xue A

Abstract
Background : Mutations in tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key player in the negative feedback regulation of nuclear factor-κB signaling, have recently been recognized as leading to early onset autoinflammatory and autoimmune syndrome. Here, we have reported the phenotypes of 3 infantile onset intractable inflammatory bowel disease (IBD) patients with TNFAIP3 mutations and reviewed previously reported cases to establish phenotypic features associated with TNFAIP3 monogenicity.
Methods: From January 1, 2015, to December 31, 2017, we recruited 58 infantile-onset IBD patients. Targeted sequencing and whole-exome sequencing were performed. Sanger sequencing confirmed the variants and determined the parental origin. We followed all the patients with TNFAIP3 mutations in our cohort and analyzed their clinical data.
Results: Genetic screening in all 58 patients with infantile-onset IBD revealed 44 (75.9%) cases of monogenic disorders, and 3 de novo TNFAIP3 mutations were identified, including 1 nonsense and 2 frame shift mutations. All the mutations resulted in premature stop codon. All 3 patients had multiple systemic involvements, with predominant gastrointestinal diseases.
Conclusions: Most infantile-onset IBD was associated with monogenetic mutation, and in addition to the 50 reported genes, other rare genetic variants need to be determined. TNFAIP3 may be an important candidate gene. The treatment of TNFAIP3-associated infantile-onset-IBD was challenging.

PMID: 29788367 [PubMed - as supplied by publisher]

Nonsynonymous Polymorphism in Guanine Monophosphate Synthetase Is a Risk Factor for Unfavorable Thiopurine Metabolite Ratios in Patients With Inflammatory Bowel Disease.

Inflamm Bowel Dis. 2018 May 16;:

Authors: Roberts RL, Wallace MC, Seinen ML, van Bodegraven AA, Krishnaprasad K, Jones GT, van Rij AM, Baird A, Lawrance IC, Prosser R, Bampton P, Grafton R, Simms LA, Studd C, Bell SJ, Kennedy MA, Halliwell J, Gearry RB, Radford-Smith G, Andrews JM, McHugh PC, Barclay ML

Abstract
Background : Up to 20% of patients with inflammatory bowel disease (IBD) who are refractory to thiopurine therapy preferentially produce 6-methylmercaptopurine (6-MMP) at the expense of 6-thioguanine nucleotides (6-TGN), resulting in a high 6-MMP:6-TGN ratio (>20). The objective of this study was to evaluate whether genetic variability in guanine monophosphate synthetase (GMPS) contributes to preferential 6-MMP metabolizer phenotype.
Methods: Exome sequencing was performed in a cohort of IBD patients with 6-MMP:6-TGN ratios of >100 to identify nonsynonymous single nucleotide polymorphisms (nsSNPs). In vitro assays were performed to measure GMPS activity associated with these nsSNPs. Frequency of the nsSNPs was measured in a cohort of 530 Caucasian IBD patients.
Results: Two nsSNPs in GMPS (rs747629729, rs61750370) were detected in 11 patients with very high 6-MMP:6-TGN ratios. The 2 nsSNPs were predicted to be damaging by in silico analysis. In vitro assays demonstrated that both nsSNPs resulted in a significant reduction in GMPS activity (P < 0.05). The SNP rs61750370 was significantly associated with 6-MMP:6-TGN ratios ≥100 (odds ratio, 5.64; 95% confidence interval, 1.01-25.12; P < 0.031) in a subset of 264 Caucasian IBD patients.
Conclusions: The GMPS SNP rs61750370 may be a reliable risk factor for extreme 6MMP preferential metabolism.

PMID: 29788244 [PubMed - as supplied by publisher]

Diagnostic Yield of Next-Generation Sequencing in Very Early-Onset Inflammatory Bowel Diseases: A Multicenter Study.

J Crohns Colitis. 2018 May 18;:

Authors: Charbit-Henrion F, Parlato M, Hanein S, Duclaux-Loras R, Nowak J, Begue B, Rakotobe S, Bruneau J, Fourrage C, Alibeu O, Rieux-Laucat F, Lévy E, Stolzenberg MC, Mazerolles F, Latour S, Lenoir C, Fischer A, Picard C, Aloi M, Amil Dias J, Ben Hariz M, Bourrier A, Breuer C, Breton A, Bronski J, Buderus S, Cananzi M, Coopman S, Crémilleux C, Dabadie A, Dumant-Forest C, Egritas Gurkan O, Fabre A, Fischer A, German Diaz M, Gonzalez-Lama Y, Goulet O, Guariso G, Gurcan N, Homan M, Hugot JP, Jeziorski E, Karanika E, Lachaux A, Lewindon P, Lima R, Magro F, Major J, Malamut G, Mas E, Mattyus I, Mearin LM, Melek J, Navas-Lopez VM, Paerregaard A, Pelatan C, Pigneur B, Pinto Pais I, Rebeuh J, Romano C, Siala N, Strisciuglio C, Tempia-Caliera M, Tounian P, Turner D, Urbonas V, Willot S, Ruemmele FM, Cerf-Bensussan N

Abstract
Background and Aims: An expanding number of monogenic defects have been identified as causative of severe forms of very early-onset inflammatory bowel diseases (VEO-IBD). The present study aimed at defining how next-generation sequencing (NGS) methods can be used to improve identification of known molecular diagnosis and adapt treatment.
Methods: 207 children were recruited in 45 Paediatric centres through an international collaborative network (ESPGHAN GENIUS working group) with a clinical presentation of severe VEO-IBD (n=185) or an anamnesis suggestive of a monogenic disorder (n=22). Patients were divided at inclusion into three phenotypic subsets: predominantly small bowel inflammation, colitis with perianal lesions, and colitis only. Methods to obtain molecular diagnosis included functional tests followed by specific Sanger sequencing, custom-made targeted NGS, and in selected cases whole exome sequencing (WES) of parents-child trios. Genetic findings were validated clinically and/or functionally.
Results: Molecular diagnosis was achieved in 66/207 children (32%): 61% with small bowel inflammation, 39% with colitis and perianal lesions and 18% with colitis only. Targeted NGS pinpointed gene mutations causative of atypical presentations and identified large exonic copy number variations previously missed by WES.
Conclusions: Our results lead us to propose an optimised diagnostic strategy to identify known monogenic causes of severe IBD.

PMID: 29788237 [PubMed - as supplied by publisher]

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

Mol Metab. 2018 May 16;:

Authors: Montagne L, Derhourhi M, Piton A, Toussaint B, Durand E, Vaillant E, Thuillier D, Gaget S, De Graeve F, Rabearivelo I, Lansiaux A, Lenne B, Sukno S, Desailloud R, Cnop M, Nicolescu R, Cohen L, Zagury JF, Amouyal M, Weill J, Muller J, Sand O, Delobel B, Froguel P, Bonnefond A

Abstract
OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step.
METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145).
RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations.
CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

PMID: 29784605 [PubMed - as supplied by publisher]

A homozygous loss-of-function CAMK2A mutation causes growth delay, frequent seizures and severe intellectual disability.

Elife. 2018 May 22;7:

Authors: Chia PH, Zhong FL, Niwa S, Bonnard C, Utami KH, Zeng R, Lee H, Eskin A, Nelson SF, Xie WH, Al-Tawalbeh S, El-Khateeb M, Shboul M, Pouladi MA, Al-Raqad M, Reversade B

Abstract
Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

PMID: 29784083 [PubMed - in process]

Severe hepatopathy and neurological deterioration after start of valproate treatment in a 6-year-old child with mitochondrial tryptophanyl-tRNA synthetase deficiency.

Orphanet J Rare Dis. 2018 May 21;13(1):80

Authors: Vantroys E, Smet J, Vanlander AV, Vergult S, De Bruyne R, Roels F, Stepman H, Roeyers H, Menten B, Van Coster R

Abstract
BACKGROUND: The first subjects with deficiency of mitochondrial tryptophanyl-tRNA synthetase (WARS2) were reported in 2017. Their clinical characteristics can be subdivided into three phenotypes (neonatal phenotype, severe infantile onset phenotype, Parkinson-like phenotype).
RESULTS: Here, we report on a subject who presented with early developmental delay, motor weakness and intellectual disability and who was considered during several years as having a non-progressive encephalopathy. At the age of six years, she had an epileptic seizure which was treated with sodium valproate. In the months after treatment was started, she developed acute liver failure and severe progressive encephalopathy. Although valproate was discontinued, she died six months later. Spectrophotometric analysis of the oxidative phosphorylation complexes in liver revealed a deficient activity of complex III and low normal activities of the complexes I and IV. Activity staining in the BN-PAGE gel confirmed the low activities of complex I, III and IV and, in addition, showed the presence of a subcomplex of complex V. Histochemically, a mosaic pattern was seen in hepatocytes after cytochrome c oxidase staining. Using Whole Exome Sequencing two known pathogenic variants were detected in WARS2 (c.797delC, p.Pro266ArgfsTer10/ c.938 A > T, p.Lys313Met).
CONCLUSION: This is the first report of severe hepatopathy in a subject with WARS2 deficiency. The hepatopathy occurred soon after start of sodium valproate treatment. In the literature, valproate-induced hepatotoxicity was reported in the subjects with pathogenic mutations in POLG and TWNK. This case report illustrates that the course of the disease in the subjects with a mitochondrial defect can be non-progressive during several years. The subject reported here was first diagnosed as having cerebral palsy. Only after a mitochondriotoxic medication was started, the disease became progressive, and the diagnosis of a mitochondrial defect was made.

PMID: 29783990 [PubMed - in process]

Identification of a mutation in CNNM4 by whole exome sequencing in an Amish family and functional link between CNNM4 and IQCB1.

Mol Genet Genomics. 2018 Jun;293(3):699-710

Authors: Li S, Xi Q, Zhang X, Yu D, Li L, Jiang Z, Chen Q, Wang QK, Traboulsi EI

Abstract
We investigated an Amish family in which three siblings presented with an early-onset childhood retinal dystrophy inherited in an autosomal recessive fashion. Genome-wide linkage analysis identified significant linkage to marker D2S2216 on 2q11 with a two-point LOD score of 1.95 and a multi-point LOD score of 3.76. Whole exome sequencing was then performed for the three affected individuals and identified a homozygous nonsense mutation (c.C1813T, p.R605X) in the cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) gene located within the 2p14-2q14 Jalili syndrome locus. The initial assessment and collection of the family were performed before the clinical delineation of Jalili syndrome. Another assessment was made after the discovery of the responsible gene and the dental abnormalities characteristic of Jalili syndrome were retrospectively identified. The p.R605X mutation represents the first probable founder mutation of Jalili syndrome identified in the Amish community. The molecular mechanism underlying Jalili syndrome is unknown. Here we show that CNNM4 interacts with IQCB1, which causes Leber congenital amaurosis (LCA) when mutated. A truncated CNNM4 protein starting at R605 significantly increased the rate of apoptosis, and significantly increased the interaction between CNNM4 and IQCB1. Mutation p.R605X may cause Jalili syndrome by a nonsense-mediated decay mechanism, affecting the function of IQCB1 and apoptosis, or both. Our data, for the first time, functionally link Jalili syndrome gene CNNM4 to LCA gene IQCB1, providing important insights into the molecular pathogenic mechanism of retinal dystrophy in Jalili syndrome.

PMID: 29322253 [PubMed - indexed for MEDLINE]

TCR Repertoire Intratumor Heterogeneity in Localized Lung Adenocarcinomas: An Association with Predicted Neoantigen Heterogeneity and Postsurgical Recurrence.

Cancer Discov. 2017 10;7(10):1088-1097

Authors: Reuben A, Gittelman R, Gao J, Zhang J, Yusko EC, Wu CJ, Emerson R, Zhang J, Tipton C, Li J, Quek K, Gopalakrishnan V, Chen R, Vence LM, Cascone T, Vignali M, Fujimoto J, Rodriguez-Canales J, Parra ER, Little LD, Gumbs C, Forget MA, Federico L, Haymaker C, Behrens C, Benzeno S, Bernatchez C, Sepesi B, Gibbons DL, Wargo JA, William WN, Swisher S, Heymach JV, Robins H, Lee JJ, Sharma P, Allison JP, Futreal PA, Wistuba II, Zhang J

Abstract
Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity.Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088-97. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

PMID: 28733428 [PubMed - indexed for MEDLINE]

Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy.

Eur Urol. 2017 Oct;72(4):544-554

Authors: Seiler R, Ashab HAD, Erho N, van Rhijn BWG, Winters B, Douglas J, Van Kessel KE, Fransen van de Putte EE, Sommerlad M, Wang NQ, Choeurng V, Gibb EA, Palmer-Aronsten B, Lam LL, Buerki C, Davicioni E, Sjödahl G, Kardos J, Hoadley KA, Lerner SP, McConkey DJ, Choi W, Kim WY, Kiss B, Thalmann GN, Todenhöfer T, Crabb SJ, North S, Zwarthoff EC, Boormans JL, Wright J, Dall'Era M, van der Heijden MS, Black PC

Abstract
BACKGROUND: An early report on the molecular subtyping of muscle-invasive bladder cancer (MIBC) by gene expression suggested that response to neoadjuvant chemotherapy (NAC) varies by subtype.
OBJECTIVE: To investigate the ability of molecular subtypes to predict pathological downstaging and survival after NAC.
DESIGN, SETTING, AND PARTICIPANTS: Whole transcriptome profiling was performed on pre-NAC transurethral resection specimens from 343 patients with MIBC. Samples were classified according to four published molecular subtyping methods. We developed a single-sample genomic subtyping classifier (GSC) to predict consensus subtypes (claudin-low, basal, luminal-infiltrated and luminal) with highest clinical impact in the context of NAC. Overall survival (OS) according to subtype was analyzed and compared with OS in 476 non-NAC cases (published datasets).
INTERVENTION: Gene expression analysis was used to assign subtypes.
OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Receiver-operating characteristics were used to determine the accuracy of GSC. The effect of GSC on survival was estimated by Cox proportional hazard regression models.
RESULTS AND LIMITATIONS: The models generated subtype calls in expected ratios with high concordance across subtyping methods. GSC was able to predict four consensus molecular subtypes with high accuracy (73%), and clinical significance of the predicted consensus subtypes could be validated in independent NAC and non-NAC datasets. Luminal tumors had the best OS with and without NAC. Claudin-low tumors were associated with poor OS irrespective of treatment regimen. Basal tumors showed the most improvement in OS with NAC compared with surgery alone. The main limitations of our study are its retrospective design and comparison across datasets.
CONCLUSIONS: Molecular subtyping may have an impact on patient benefit to NAC. If validated in additional studies, our results suggest that patients with basal tumors should be prioritized for NAC. We discovered the first single-sample classifier to subtype MIBC, which may be suitable for integration into routine clinical practice.
PATIENT SUMMARY: Different molecular subtypes can be identified in muscle-invasive bladder cancer. Although cisplatin-based neoadjuvant chemotherapy improves patient outcomes, we identified that the benefit is highest in patients with basal tumors. Our newly discovered classifier can identify these molecular subtypes in a single patient and could be integrated into routine clinical practice after further validation.

PMID: 28390739 [PubMed - indexed for MEDLINE]

Identification of mutations through dominant screening for obesity using C57BL/6 substrains.

Sci Rep. 2016 09 02;6:32453

Authors: Hossain MS, Asano F, Fujiyama T, Miyoshi C, Sato M, Ikkyu A, Kanno S, Hotta N, Kakizaki M, Honda T, Kim SJ, Komiya H, Miura I, Suzuki T, Kobayashi K, Kaneda H, Kumar V, Takahashi JS, Wakana S, Funato H, Yanagisawa M

Abstract
The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.

PMID: 27585985 [PubMed - indexed for MEDLINE]