Exome sequencing in developmental eye disease leads to identification of causal variants in GJA8, CRYGC, PAX6 and CYP1B1

Developmental eye diseases, including cataract/microcornea, Peters anomaly and coloboma/microphthalmia/anophthalmia, are caused by mutations encoding many different signalling and structural proteins in the developing eye. All modes of Mendelian inheritance occur and many are sporadic cases, so provision of accurate recurrence risk information for families and affected individuals is highly challenging. Extreme genetic heterogeneity renders testing for all known disease genes clinically unavailable with traditional methods. We used whole-exome sequencing in 11 unrelated developmental eye disease patients, as it provides a strategy for assessment of multiple disease genes simultaneously. We identified five causative variants in four patients in four different disease genes, GJA8, CRYGC, PAX6 and CYP1B1. This detection rate (36%) is high for a group of patients where clinical testing is frequently not undertaken due to lack of availability and cost. The results affected clinical management in all cases. These variants were detected in the cataract/microcornea and Peters anomaly patients. In two patients with coloboma/microphthalmia, variants in ABCB6 and GDF3 were identified with incomplete penetrance, highlighting the complex inheritance pattern associated with this phenotype. In the coloboma/microphthalmia patients, four other variants were identified in CYP1B1, and CYP1B1 emerged as a candidate gene to be considered as a modifier in coloboma/microphthalmia

Value of whole exome sequencing for syndromic retinal dystrophy diagnosis in young patients

Background: Several retinal dystrophies are associated with syndromic features including such conditions as Bardet–Biedl and Joubert syndromes. Cohen syndrome is an autosomal recessive disorder associated with multiple clinical manifestations including developmental delay, acquired microcephaly, myopia, pigmentary retinopathy, joint hypermobility, truncal obesity, friendly disposition and intermittent neutropenia. In young patients, diagnosis is difficult, because several of the characteristic features may not be present until school age or later years and the intermittent neutropenia is not always detectable. Design: This was a prospective study using whole exome sequencing in syndromic retinal dystrophy. It was undertaken in a hospital and research institute setting. Participants: Participants in this study were members of a consanguineous Australian family of Lebanese ethnicity with two siblings with retinal dystrophy, microcephaly and developmental delay. Methods: Detailed clinical evaluation was undertaken. Whole exome capture and sequencing of patient genomic DNA samples was followed by sequence alignment, variant detection, comparison and prioritization. Main Outcome Measures: Pathogenic variant identification in the disease-causing gene in affected individuals. Results: We identified a novel homozygous deletion leading to a frameshift mutation in VPS13B, c.11327del, p.(Asn3776Thrfs*102), the disease gene associated with Cohen syndrome. Conclusions: This report emphasizes the value of a broad-based whole exome sequencing approach in disease gene identification in the syndromic retinal dystrophies, where all disease characteristics may not be present in young patients to allow a clinical diagnosis. This facilitates improved prognostic and genetic information for patients and families.7 page(s

Advantage of Whole Exome Sequencing over Allele-specific and Targeted Segment Sequencing, in Detection of Novel <i>TULP1</i> Mutation in Leber Congenital Amaurosis

<div><p></p><p><i>Background</i>: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.</p><p><i>Materials and Methods</i>: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.</p><p><i>Results</i>: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C > T, p.Arg361*) in <i>TULP1</i>, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.</p><p><i>Conclusions</i>: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.</p></div

Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46, XY, t(2;19)(q37.3; q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans

International audienceBackground Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6 , which have been previously associated with this disorder. Methods We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants. Results We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures. Conclusion Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT–β-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development

TMD Handbook

471 pages, many figuresThis handbook provides a comprehensive review of transverse-momentum-dependent parton distribution functions and fragmentation functions, commonly referred to as transverse momentum distributions (TMDs). TMDs describe the distribution of partons inside the proton and other hadrons with respect to both their longitudinal and transverse momenta. They provide unique insight into the internal momentum and spin structure of hadrons, and are a key ingredient in the description of many collider physics cross sections. Understanding TMDs requires a combination of theoretical techniques from quantum field theory, nonperturbative calculations using lattice QCD, and phenomenological analysis of experimental data. The handbook covers a wide range of topics, from theoretical foundations to experimental analyses, as well as recent developments and future directions. It is intended to provide an essential reference for researchers and graduate students interested in understanding the structure of hadrons and the dynamics of partons in high energy collisions