Congenital Short Bowel Syndrome: from clinical and genetic diagnosis to the molecular mechanisms involved in intestinal elongation

AbstractCongenital Short Bowel Syndrome (CSBS) is a rare gastrointestinal disorder in which the mean length of the small intestine is substantially reduced when compared to its normal counterpart. Families with several affected members have been described and CSBS has been suggested to have a genetic basis. Recently, our group found mutations in CLMP as the cause of the recessive form of CSBS, and mutations in FLNA as the cause of the X-linked form of the disease. These findings have improved the quality of genetic counselling for CSBS patients and made prenatal diagnostics possible. Moreover, they provided a reliable starting point to further investigate the pathogenesis of CSBS, and to better understand the development of the small intestine. In this review, we present our current knowledge on CSBS and discuss hypotheses on how the recent genetic findings can help understand the cause of CSBS

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder

Congenital short bowel syndrome as the presenting symptom in male patients with FLNA mutations

<p>Purpose: Autosomal recessive congenital short bowel syndrome is caused by mutations in CLMP. No mutations were found in the affected males of a family with presumed X-linked congenital short bowel syndrome or in an isolated male patient. Our aim was to identify the disease-causing mutation in these patients.</p><p>Methods: We performed mutation analysis of the second exon of FLNA in the two surviving affected males of the presumed X-linked family and in the isolated patient.</p><p>Results: We identified a novel 2-base-pair deletion in the second exon of FLNA in all these male patients. The deletion is located between two nearby methionines at the N-terminus of filamin A. Previous studies showed that translation of FLNA occurs from both methionines, resulting in two isoforms of the protein. We hypothesized that the longer isoform is no longer translated due to the mutation and that this mutation is therefore not lethal for males in utero.</p><p>Conclusion: Our findings emphasize that congenital short bowel syndrome can be the presenting symptom in male patients with mutations in FLNA. Genet Med 2013:15(4):310-313</p>

Diagnostic exome sequencing in 266 Dutch patients with visual impairment

Inherited eye disorders have a large clinical and genetic heterogeneity, which makes genetic diagnosis cumbersome. An exome-sequencing approach was developed in which data analysis was divided into two steps: the vision gene panel and exome analysis. In the vision gene panel analysis, variants in genes known to cause inherited eye disorders were assessed for pathogenicity. If no causative variants were detected and when the patient consented, the entire exome data was analyzed. A total of 266 Dutch patients with different types of inherited eye disorders, including inherited retinal dystrophies, cataract, developmental eye disorders and optic atrophy, were investigated. In the vision gene panel analysis (likely), causative variants were detected in 49% and in the exome analysis in an additional 2% of the patients. The highest detection rate of (likely) causative variants was in patients with inherited retinal dystrophies, for instance a yield of 63% in patients with retinitis pigmentosa. In patients with developmental eye defects, cataract and optic atrophy, the detection rate was 50, 33 and 17%, respectively. An exome-sequencing approach enables a genetic diagnosis in patients with different types of inherited eye disorders using one test. The exome approach has the same detection rate as targeted panel sequencing tests, but offers a number of advantages. For instance, the vision gene panel can be frequently and easily updated with additional (novel) eye disorder genes. Determination of the genetic diagnosis improved the clinical diagnosis, regarding the assessment of the inheritance pattern as well as future disease perspective

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

We thank Ellen Blokland, Lonneke Duijkers, Duaa Elmelik, Anita Hoogendoorn, Marlie Jacobs-Camps, Saskia van der Velde-Visser, and Marijke Zonneveld-Vrieling for technical assistance. We thank Sabine Defoort, Hélène Dollfus, Isabelle Drumare, Christian P. Hamel, Karsten Hufendiek, Cord Huchzermeyer, Herbert Jägle, Ulrich Kellner, Philipp Rating, Klaus Rüther, Eric Souied, Georg Spital, and Xavier Zanlonghi for their cooperation and ascertaining STGD1 cases. This work was supported by the FP7-PEOPLE-2012-ITN programme EyeTN, agreement 317472 (to F.P.M.C.); the Macula Vision Research Foundation (to F.P.M.C.); the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD (to A.G., C.B.H., F.P.M.C., R.W.J.C., and S.A.); the RP Fighting Blindness UK (RetinaUK), grant no. GR591 (to F.P.M.C. and S.A.); the Rotterdamse Stichting Blindenbelangen, the Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C. and S.A.); and by the Landelijke Stichting voor Blinden en Slechtzienden, Macula Degeneratie fonds, and the Stichting Blinden-Penning, which contributed through Uitzicht 2016-12 (to F.P.M.C. and S.A.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds, which contributed through UitZicht 2015-31, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo (to A.G. and R.W.J.C.); the Stichting Macula Degeneratie Fonds; and the Stichting A.F. Deutman Researchfonds Oogheelkunde (to C.B.H.). This work was also supported by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and Landelijke Stichting voor Blinden en Slechtzienden, which contributed through UitZicht 2014-13, together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, and the Stichting tot Verbetering van het Lot der Blinden (to F.P.M.C.), the Ghent University Research Fund (BOF15/GOA/011), the Research Foundation Flanders (FVO) G0C6715N, and the Hercules Foundation AUGE/13/023 and JED Foundation to E.D.B. M.B. was PhD fellow of the FWO and recipient of a grant of the funds for Research in Ophthalmology (FRO). E.D.B. is Senior Clinical Investigator of the FWO (1802215N). This work was also supported by the National Institute for Health Research (NIHR) Biomedical Centre at Moorfields and UCL Institute of Ophthalmology (to A.W.), UK NIHR Rare Disease Translational Research Consortium (to G.A. and A.W.), NIHR for the NIHR BioResource (RG65966) (to F.L.R.) by grants from the Federal Ministry of Education and Research (BMBF) (ref. IDs 01GM0851 and 01GM1108B) (to B.H.F.W.). G.A. is supported by a Fight for Sight UK Early Career Investigator Award. The funding organizations had no role in the design or conduct of this research, and provided unrestricted grants. This study made use of data generated by the Genome of the Netherlands Project. Funding for the project was provided by the Netherlands Organization for Scientific Research under award number 184021007, dated 9 July 2009 and made available as a Rainbow Project of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL). Samples where contributed by LifeLines (http://lifelines.nl/lifelines-research/general), the Leiden Longevity Study (http://www.healthy-ageing.nl; http://www.langleven.net), the Netherlands Twin Registry (NTR: http://www.tweelingenregister.org), the Rotterdam studies (http://www.erasmus-epidemiology.nl/rotterdamstudy), and the Genetic Research in Isolated Populations program (http://www.epib.nl/research/geneticepi/research.html#gip). The sequencing was carried out in collaboration with the Beijing Institute for Genomics (BGI).Peer reviewedPublisher PD

Differential Contributions of Rare and Common, Coding and Noncoding Ret Mutations to Multifactorial Hirschsprung Disease Liability

The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ubiquity of a >4-fold susceptibility from a C→T allele (rs2435357: p = 3.9 × 10−43 in European ancestry; p = 1.1 × 10−21 in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%–30%/47% and case frequency of 54%–62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 × 10−5) and familiality (p = 6.2 × 10−4). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a “cellular-recessive” genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied