Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids

Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro

Association of a germline copy number polymorphism of APOBEC3A and APOBEC3B with burden of putative APOBEC-dependent mutations in breast cancer.

The somatic mutations in a cancer genome are the aggregate outcome of one or more mutational processes operative through the lifetime of the individual with cancer. Each mutational process leaves a characteristic mutational signature determined by the mechanisms of DNA damage and repair that constitute it. A role was recently proposed for the APOBEC family of cytidine deaminases in generating particular genome-wide mutational signatures and a signature of localized hypermutation called kataegis. A germline copy number polymorphism involving APOBEC3A and APOBEC3B, which effectively deletes APOBEC3B, has been associated with modestly increased risk of breast cancer. Here we show that breast cancers in carriers of the deletion show more mutations of the putative APOBEC-dependent genome-wide signatures than cancers in non-carriers. The results suggest that the APOBEC3A-APOBEC3B germline deletion allele confers cancer susceptibility through increased activity of APOBEC-dependent mutational processes, although the mechanism by which this increase in activity occurs remains unknown.We would like to thank the Wellcome Trust for support (grant reference 098051). SN-Z is a Wellcome-Beit Prize Fellow and is supported through a Wellcome Trust Intermediate Fellowship (grant reference WT100183MA). PJC is personally funded through a Wellcome Trust Senior Clinical Research Fellowship (grant reference WT088340MA). NB is an EHA fellow and is supported by a Lady Tata Memorial Trust award. The H.L. Holmes Award from the National Research Council Canada and an EMBO Fellowship supports AS

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

The search for tourette syndrome genes : a conceptual and experimental approach

Dissertation (PhD) -- University of Stellenbosch, 1999.ENGLISH SUMMARY: Tourette syndrome has been reported in most populations throughout the world. Overall, there appears to be similar clinical phenomenology and psychopathology, which may serve as an indication of the biological nature for the condition. The diagnosis of Tourette syndrome represents a challenge for physicians because of clinical heterogeneity and often-present comorbidity with other known neurobehavioural conditions. Due to these clinical overlaps Tourette syndrome may serve as a model disorder for investigating the relationship between various neurological and behavioral domains of childhood reflecting either the expression of a common biological pathway or a common genetic background. The understanding of the genetic basis of Tourette syndrome is therefore of special importance, because it may provide useful insights for the study of other developmental disorders. However, the lack of objective biological markers of clinical manifestation together with a possible high phenocopy rate, unclear mode of inheritance, incomplete penetrance, and frequent bilinear transmission of predisposing genes represent major obstacles for those attempting to elucidate the genetic basis of Tourette syndrome. The research presented in this document is a result of six years' effort of the author and her collaborators to generate cytogenetic and molecular genetic data contributing to a better understanding of genetic and environmental factors affecting the phenotypic expression of Tourette syndrome. Theoretical and experimental results of this collaborative effort are assembled in seven articles (four published, three currently submitted for a publication) and a general introductory section relating to the problems, methods and methodology described and utilized in data collection for the individual papers. Taken as a whole, while the study of chromosome fragile site expression in Tourette syndrome probands yielded equivocal results leading to a number of rather speculative but interesting interpretations, the results of subsequent molecular genetic studies are far clearer. The three most valuable outcomes of these studies for future genetic investigations in Tourette syndrome gene-mapping efforts in the Afrikaner population, and complex genetic traits in general, are: I. The evidence for association/linkage of at least three genomic regions with Tourette syndrome in the Afrikaner population, with two of the regions (11q23 and 8q22) being suggestively linked to Tourette syndrome by others in different populations and employing different analytical methods. 2. The evidence for extended background linkage disequilibrium in the general Afrikaner population (> 5 cM) which further strengthens existing experimental data demonstrating the suitability of this population for gene-mapping efforts involving complex traits. 3. The proof based on real rather than computer-simulated data that sequential and semiparametric methods of analysis could be sufficiently powerful to generate cumulative evidence for positive linkage with the trait in the regions which repeatedly yielded both highly significant as well as suggestively significant disease-marker associations in the initial set of samples.AFRIKAANSE OPSOMMING: Tourettesindroom is 'n algemene oorerflike neurobiologiese probleem wat in verskeie bevolkingsgroepe vanoor die wereld beskryf is. As gevolg van identiese fenomenologie en psigopatologie ten spyte van omgewingsverskille, is dit aanduidend van 'n sterk biologiese grondslag vir die toe stand. Die teenwoordigheid van kliniese meersoortigheid en die verhoogde voorkoms van 'n verskeidenheid komorbiede probleme by 'n subgroep van individue met Tourettesindroom, veroorsaak dikwels probleme met die akkurate identifisering hiervan. Dit skep egter ook geleenthede vir die bestudering by kinders, van verskeie neurologiese en gedragsmanifestasies gebaseer op 'n gemene genetiese substraat. Insig in die genetiese-omgewings wisselwerking by Tourettesindroom baan dus die weg vir begrip van ander ontwikkelingsprobleme wat ook by kinders aangetref word. Die afwesigheid van 'n betroubare biologiese merker of merkers vir hierdie kliniese entiteit, die algemene voorkoms van fenokopiee, komplekse oorerwingspatroon, onvolledige penetrasie en algemene verskynsel van oorerwing vanaf beide ouers, verteenwoordig 'n aantal formidabele struikelblokke ten opsigte van die analise van die genetiese basis van Tourettesindroom. TS word as een van die komplekse oorerflike toestande beskou, wat beteken dat daar duidelike oorerflike faktore by betrokke is, maar dat die oorerwing nie-mendelies van aard is. Die gebruiklike reduksionistiese benaderings wat so suksesvol was vir die analise van die enkelgeentoestande, werk nie meer onder hierdie omstandighedenie, en vir die rede word verskeie nie-parametriese of semiparametriese modelle ingespan. Die gedokumenteerde resultate verteenwoordig die navorsing uitgevoer tesame met plaaslike en oorsese medewerkers op hierdie gebied gedurende die laaste ses jaar. Die teoretiese en eksperimentele resultate word weergegee in sewe publikasies. Hiertydens is sitogenetiese en molekulere gegewens versamel in 'n poging om die genetiese en omgewingsfaktore onderliggend tot die ekspressie van Tourettesindroom te bepaal. Die teoretiese en eksperimentele resultate van hierdie poging word weergegee in sewe publikasies, waarvan vier reeds gepubliseer is, en 'n algemene inleidende afdeling wat die probleme en metodes bespreek soos tydens die versameling en analise van die data ervaar is. Die resultate word in twee afdelings aangebied: eerstens is daar die teoretisering ten opsigte van die bevinding van chromosomale breekbaarheid, wat aangedui is om verhoog te wees in die Tourette groep. Die betekenis van hierdie bevinding is tans nog onduidelik, en as gevolg van resolusieverskille nie direk met die DNA bevindings korreleerbaar nie. Hierdie merkerareas moet egter deurgaans in gedagte gehou word as moontlik aanwysend van die ligging van kandidaatgene vir Tourettesindroom. Die belangrikste gedeelte behandel egter die benadering tot die totale genoomsifiing, sowel as die veilgheidsmaatreels ingebou deur die heranalise van verskeie subgroepe en gevolglike replisering van resultate. Die mees waardevolle implikasies van hierdie navorsing ten opsigte van die uitstippeling van die pad vorentoe vir Tourettesindroom geenkartering by die Afrikaner, en komplekse oorerflike toestande in die algemeen, sluit die volgende in: 1. Die bewyse gevind vir die bevestiging van 3 genomiese streke soos oorspronklik deur die eerste fase assosiasiestudies aangetoon by die manifestering van Tourettesindroom in die Afrikaner, en waar ten minste twee van die gebiede (11q23 en 8q22) ook deur ander navorsers in ander bevolkingsgroepe met hierdie toestand gekoppel is; 2. Die kwantifisering van die stand van koppelings-disekwilibrium by 'n aantal lokusse in die Afrikaner genepoel van < 5cM. Hierdie gegewens versterk die gedagtes met betrekking tot die geskiktheid van hierdie bevolkingsgroep vir geenkarteringspogings vir komplekse toestande; 3. Die bewys, gebaseer op reele in stede van gemodelleerde data, dat opeenvolgende, semiparametriese analisemetodes oor voldoende statistiese krag beskik om kumulatiewe getuienis te verskaf vir positiewe koppeling van TS met streke wat ook in die oorspronkilke siektemerker assosiasiestudies betekenisvolle resultate gelewer het

Novel deletion variants of 9q13–q21.12 and classical euchromatic variants of 9q12/qh involve deletion, duplication and triplication of large tracts of segmentally duplicated pericentromeric euchromatin

Large-scale copy number variation that is cytogenetically visible in normal individuals has been described as euchromatic variation but needs to be distinguished from pathogenic euchromatic deletion or duplication. Here, we report eight patients (three families and two individuals) with interstitial deletions of 9q13-q21.12. Fluorescence in situ hybridisation with a large panel of BACs showed that all the deleted clones were from extensive tracts of segmentally duplicated euchromatin, copies of which map to both the long and short arms of chromosome 9. The variety of reasons for which these patients were ascertained, and the phenotypically normal parents, indicates that this is a novel euchromatic variant with no phenotypic effect. Further, four patients with classical euchromatic variants of 9q12/qh or 9p12 were also shown to have duplications or triplications of this segmentally duplicated material common to both 9p and 9q. The cytogenetic boundaries between the segmentally duplicated regions and flanking unique sequences were mapped to 9p13.1 in the short arm (BAC RP11-402N8 at 38.7 Mb) and to 9q21.12 in the long arm (BAC RP11-88I18 at 70.3 Mb). The BACs identified in this study should in future make it possible to differentiate between clinically significant deletions or duplications and euchromatic variants with no established phenotypic consequence