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Background Bazex–Dupré–Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11·4-Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. Objectives To investigate the genetic aetiology and molecular mechanisms underlying BDCS. Methods We ascertained multiple individuals from eight unrelated families affected with BDCS (F1–F8). Whole-exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array comparative genomic hybridization and quantitative polymerase chain reaction (PCR) were used to explore copy number variations, followed by long-range gap PCR and Sanger sequencing to amplify the duplication junctions and to define the head–tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from patients with BDCS and sporadic BCCs. The ACTRT1 variant c.547dup (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with t allele frequency calculator. Results In eight families with BDCS, we identified overlapping 18–135-kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed that the duplications did not affect the topologically associated domain, but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cell compartment, and found increased ARHGAP36 levels in hair follicles in telogen, in BCCs and in trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted maximum tolerated minor allele frequency of ACTRT1 variants in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss of function of ACTRT1 variants to be an unlikely cause for BDCS. Conclusions Noncoding Xq26.1 duplications cause BDCS. The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs. What is already known about this topic? Bazex–Dupré–Christol syndrome (BDCS) is a rare X-linked basal cell carcinoma susceptibility syndrome linked to an 11·4-Mb interval on chromosome Xq25-q27.1.Loss-of-function variants in ACTRT1 and its regulatory elements were suggested to cause BDCS. What does this study add? BDCS is caused by small tandem noncoding intergenic duplications at chromosome Xq26.1.The Xq26.1 BDCS duplications likely dysregulate ARHGAP36, the flanking centromeric gene.ACTRT1 loss-of-function variants are unlikely to cause BDCS. What is the translational message? This study provides the basis for accurate genetic testing for BDCS, which will aid precise diagnosis and appropriate surveillance and clinical management.ARHGAP36 may be a novel therapeutic target for all forms of sporadic basal cell carcinomas.

Urinary tract malformations account for half of all children with kidney failure, and some have defined monogenic causes. One such disorder is urofacial, or Ochoa, syndrome (UFS), an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. Incomplete voiding predisposes to urosepsis and kidney failure. Half of individuals with UFS carry biallelic variants inHPSE2, whereas some carry variants inLRIG2(leucine rich repeats and immunoglobulin like domains 2). We report one new kindred where the index case presented with fetal hydronephrosis and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with urinary tract disease, characterizes UFS. WhileHPSE2sequencing was normal, he carried a homozygous, predicted pathogenic, stop variant (c.1939C>T; p.Arg647*) inLRIG2. Hypothesizing that neurogenic defects underlieLRIG2-associated bladder dysfunction, we studiedLrig2homozygous mutant mice. Juveniles had enlarged bladders andex vivophysiology experiments showed neurogenic defects in outflow tract relaxation. Mutants also displayed abnormal detrusor contractility. Moreover, there were nuanced differences in physiological defects between the sexes. The current case emphasizes that urinary tract disease in UFS begins before birth. Putting this family in the context of all reported urinary tract disease-associatedLRIG2variants, the urinary and facial phenotype of UFS occurs with biallelic putative loss of function variants, but missense variants lead to bladder-limited disease without the grimace. Finally, our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in

BackgroundBazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11.4 Mb interval on chromosome Xq25-27.1. However, the genetic mechanism of BDCS remains an open question.MethodsTo investigate the genetic etiology of BDCS, we ascertained eight families with individuals affected with BDCS (F1-F8). Whole exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array-comparative genomic hybridization and quantitative PCR were used to explore copy number variations (CNV) in BDCS families, followed by long-range gap-PCR and Sanger sequencing to amplify duplication junction and define the precise head-tail junctions, respectively. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from BDCS patients and sporadic BCCs to detect the expression of corresponding genes. The ACTRT1 variant (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with allele frequency calculator.ResultsIn eight BDCS families, we identified overlapping 18-135kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. We detected ARHGAP36 expression near the control hair follicular stem cells compartment, and found increased ARHGAP36 levels in hair follicles in telogen, BCCs and trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted ACTRT1 variants maximum tolerated minor allele frequency in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss-of-function of ACTRT1 is unlikely to cause BDCS.ConclusionsOur data support the pathogenicity of intergenic duplications at Xq26.1, most likely leading to dysregulation of ARHGAP36, establish BDCS as a genomic disorder, and provide a potential therapeutic target for both inherited and sporadic BCCs.

Bladder exstrophy-epispadias complex (BEEC) comprises a spectrum of anterior midline congenital malformations, involving the lower urinary tract. BEEC is usually sporadic, but families with more than one affected member have been reported, and a twin concordance study supported a genetic contribution to pathogenesis. Moreover, diverse chromosomal aberrations have been reported in a small subset of individuals with BEEC. The commonest are 22q11.2 microduplications, identified in approximately 3% of BEEC index cases.We aimed to refine the chromosome 22q11.2 locus, and to determine whether the encompassed genes are expressed in normal developing and mature human urinary bladders.Using DNA from an individual with CBE, the 22q11.2 duplicated locus was refined by identification of a maternally inherited 314 kb duplication (chr22:21,147,293-21,461,017), as depicted in this image. Moreover, the eight protein coding genes within the locus were found to be expressed during normal developing and mature bladders. To determine whether duplications in any of these individual genes were associated with CBE, we undertook copy number analyses in 115 individuals with CBE without duplications of the whole locus. No duplications of individual genes were found.The current study has refined the 22q11.2 locus associated with BEEC and has shown that the eight protein coding genes are expressed in human bladders both during antenatal development and postnatally. Nevertheless, the precise biological explanation as to why duplication of the phenocritical region of 22q11 confers increased susceptibility to BEEC remains to be determined. The fact that individuals with CBE without duplications of the whole locus also lacked duplication of any of the individual genes suggests that in individuals with BEEC and duplication of the 22q11.2 locus altered dosage of more than one gene may be important in BEEC etiology.The study has refined the 22q11.2 locus associated with BEEC and has shown that the eight protein coding genes within this locus are expressed in human bladders.

The developmental disorder Burn-McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre-messenger RNA splicing factor TXNL4A. Most patients have a loss-of-function variant in trans with a 34-base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs*21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258-3C>G) and a type 1 Δ34 promoter deletion. We show the c.258-3C>G variant, and a previously reported c.258-2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22bp repeated motif could be relevant to BMKS etiology. Finally, our data emphasizes the need to analyse the non-coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation.

Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains unknown. We performed a sequencing-based genome-wide association study (seqGWAS) in 132 unrelated male PUV cases and 23,727 controls of diverse ancestry, identifying statistically significant associations with common variants at 12q24.21 (p=7.8 × 10

Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains largely unknown. We analyzed whole-genome sequencing (WGS) data from 132 unrelated PUV cases and 23,727 controls of mixed ancestry and identified statistically significant associations with common variants at 12q24.21 (P=7.8x10-12; OR 0.4) and rare variants at 6p21.1 (P=2x10-8; OR 7.2), that were replicated in an independent European cohort. Bayesian fine mapping and functional annotation mapped these loci to the transcription factor TBX5 and planar cell polarity gene PTK7, respectively, with the encoded proteins detected in the normal human developing urinary tract. These findings represent the first known genetic associations of PUV, providing novel insights into the underlying biology of this poorly understood disorder and demonstrate that a mixed ancestry WGS approach can increase power for disease locus discovery and facilitate fine-mapping of causal variants.

Research question Does a genetic condition underlie the diagnosis of primary ovarian insufficiency (POI) in a 21-year-old woman with primary amenorrhoea? Design A karyotype and genetic testing for Fragile X syndrome was undertaken. A next-generation sequencing panel of 24 genes associated with syndromal and non-syndromal POI was conducted. Results A nonsense variant c.1336G>T, p.(Glu446Ter) and whole gene deletion in STAG3 were identified. Conclusions Biallelic loss of function variants in STAG3 are associated with primary ovarian failure type 8 and are a rare cause of POI.

CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly‐like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.

The bladder exstrophy-epispadias complex (BEEC) comprises of a spectrum of anterior midline defects, all affecting the lower urinary tract, the external genitalia and the bony pelvis. In extreme cases, the gastrointestinal tract is also affected. The pathogenesis of BEEC is unclear but chromosomal aberrations have been reported. In particular, duplications of 22q11.2 have been identified in eight unrelated individuals with BEEC. The current study aimed to identify chromosomal copy number variants in BEEC. Analyses was performed using the Affymetrix Genome-wide SNP6.0 assay in 92 unrelated patients cared for by two UK pediatric urology centres. Three individuals had a 22q11.2 duplication, a significantly higher number than that found in a control group of 12,500 individuals with developmental delay who had undergone microarray testing (p

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.

The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 250 variants of uncertain significance (VUSs) that underwent splicing functional analyses. It is the capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ that is likely to have the most substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; 1 in 5 of these cases could lead to new or refined diagnoses.

Ligase IV (LIG4) syndrome is a rare disorder of DNA damage repair caused by biallelic, pathogenic variants in LIG4. This is a phenotypically heterogeneous condition with clinical presentation varying from lymphoreticular malignancies in developmentally normal individuals to significant microcephaly, primordial dwarfism, radiation hypersensitivity, severe combined immunodeficiency and early mortality. Renal defects have only rarely been described as part of the ligase IV disease spectrum. We identified a consanguineous family where three siblings presenting with antenatal growth retardation, microcephaly, severe renal anomalies and skeletal abnormalities, including radial ray defects. Autozygosity mapping and exome sequencing identified a novel homozygous frameshift variant in LIG4, c.597_600delTCAG, p.(Gln200LysfsTer33), which segregated in the family. LIG4 is encoded by a single exon and so this frameshift variant is predicted to result in a protein truncated by 678 amino acids. This is the shortest predicted LIG4 protein product reported and correlates with the most severe clinical phenotype described to date. We note the clinical overlap with Fanconi anemia and suggest that LIG4 syndrome is considered in the differential diagnosis of this severe developmental disorder.

Background Hereditary alpha-tryptasemia (HAT) is a genetic trait caused by an increased alpha-tryptase tryptase alpha/beta 1 gene copy number. Basal serum mast cell tryptase (MCT) level is typically greater than or equal to 8.0 ng/mL. Objectives To study the clinical disease spectrum of HAT and determine its UK prevalence. Methods Droplet digital PCR was used to determine tryptase alpha/beta 1 copy number in 432 DNA samples from an unselected UK birth cohort and in 70 patients referred with a basal MCT level greater than 8 ng/mL. Baseline MCT concentrations and clinical presentation were also assessed in 4283 samples sent to a regional immunology laboratory. Results Duplication in alpha copy number was present in 5% of the unselected British birth cohort, with all affected individuals having a basal MCT level of greater than or equal to 8.0 ng/mL. Basal MCT levels of greater than or equal to 8.0 ng/mL were also found in 5% of the 4283 individuals referred for MCT testing because of clinical symptoms. In 70 patients confirmed to have HAT (79% with a duplication; 21% with a higher alpha gene copy number), urticaria/angioedema (51%), skin flushing (41%), food intolerances (39%), and altered bowel habits (36%) were common presenting complaints. However, clinical manifestations were not more common in patients with gene triplications or quintuplications than in those with duplications. Some immediate family members with the same genetic trait and high basal MCT levels were asymptomatic. Conclusions Five percent of people in the United Kingdom may have HAT. The diagnosis should be considered when basal MCT level is greater than or equal to 8 ng/mL. HAT has variable clinical penetrance. It may modify the expression of multifactorial allergic diseases rather than directly cause specific phenotypes.

The bladder exstrophy–epispadias complex (BEEC) is an abdominal midline malformation comprising a spectrum of congenital genitourinary abnormalities of the abdominal wall, pelvis, urinary tract, genitalia, anus, and spine. The vast majority of BEEC cases are classified as non-syndromic and the etiology of this malformation is still unknown. This review presents the current knowledge on this multifactorial disorder, including phenotypic and anatomical characterization, epidemiology, proposed developmental mechanisms, existing animal models, and implicated genetic and environmental components.

PurposeTo develop a comprehensive analysis framework to identify pre-messenger RNA splicing mutations in the context of rare disease.MethodsWe assessed ‘variants of uncertain significance’ through six in-silico prioritization strategies. Firstly, through comparison to functional analyses, we determined the precise effect on splicing of variants identified through clinical multi-disciplinary meetings. Next, we calculated the sensitivity of in-silico prioritization strategies to distinguish known splicing mutations from common variation (>2% in allele frequency in gnomAD) within relevant disease genes. These approaches defined an accurate in-silico strategy for variant prioritization, which we retrospectively applied to a large cohort of 2783 individuals who had previously received genomic testing for rare genomic disorders. We assessed the clinical impact of such prioritization strategies alongside routine diagnostic testing strategies.ResultsWe identified 21 variants that potentially impacted splicing, and used cell based splicing assays to identify those variants which disrupted normal splicing. These findings underpinned new molecular diagnoses for 14 individuals. This process established that the use of pre-defined thresholds from a machine learning splice prediction algorithm, SpliceAI, was the most efficient method for variant prioritization, with a positive predictive value of 86%. We analysed 1,346,744 variants identified through diagnostic testing for 2783 individuals and observed that splicing variant prioritization strategies would improve clarity in clinical analysis for 15% of the individuals surveyed. Prioritized variants could provide new molecular diagnoses or provide additional support for molecular diagnosis for up to 81 individuals within our cohort.ConclusionWe present an in-silico and functional analysis framework for the assessment of variants impacting pre-messenger RNA splicing which is applicable across monogenic disorders. Incorporation of these strategies improves clarity in diagnostic reporting, increases diagnostic yield and, with the advent of targeted treatment strategies, can directly alter patient clinical management.Key HighlightsWe establish an in-silico and functional analysis framework for the incorporation of splice variant assessment into diagnostic testing that is applicable across monogenic disorders.After assessment of six distinct variant prioritization strategies, we concluded that SpliceAI was the best method to accurately identify genomic variation disrupting normal pre-mRNA splicing. We determined this through (i) functional assessment of novel ‘variants of uncertain significance’ described in this study, and (ii) calculation of sensitivity and specificity for prioritization strategies to distinguish known splicing mutations from common variants in the general population.We describe novel disease-causing variants with support from cell based functional assays which underpin autosomal recessive, autosomal dominant and X-linked Mendelian disorders. This includes variants which are deeply intronic, within the nearby splice region of canonical splice sites and variants which activate cryptic splice sites within the protein-coding regions of genes.We integrated the best performing variant prioritization strategy alongside clinical diagnostic testing for 2783 individuals referred to a well-established targeted gene panel test available through the UK National Health Service. We show that integration of such strategies will increase accuracy and clarity of diagnostic reporting, including the identification of variants which could provide new diagnoses and new carrier findings for referred individuals.Functional assessment is essential for accurate clinical assessment of variants disrupting pre-mRNA splicing. We show through cell based functional assessments that variants impacting splicing may have complex impacts on pre-mRNA splicing, which may cause multiple interpretable consequences according to ACMG guidelines.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853(∗)]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

Perrault syndrome (PS) is a genetically heterogeneous disorder characterized by primary ovarian insufficiency (POI) in females and sensorineural hearing loss in males and females. In many PS subjects, causative variants have not been found in the five reported PS genes. The objective of this study was to identify the genetic cause of PS in an extended consanguineous family with six deaf individuals. Whole exome sequencing (WES) was completed on four affected members of a large family, and variants and co-segregation was confirmed by Sanger sequencing. All hearing impaired individuals, including the proband, are homozygous for a pathogenic variant of CLDN14, but this only explains the deafness. The PS proband is also homozygous for a frameshift variant (c.1453_1454delGA, p.(Glu485Lysfs*5)) in exon 7 of SGO2 encoding shugoshin 2, which is the likely cause of her concurrent ovarian insufficiency. In mouse, Sgol2a encoding shugoshin-like 2a is necessary during meiosis in both sexes to maintain the integrity of the cohesin complex that tethers sister chromatids. Human SGO2 has not previously been implicated in any disorder, but in this case of POI and perhaps others, it is a candidate for unexplained infertility.

Lissencephaly is a phenotypically and genetically heterogeneous group of cortical brain malformations due to abnormal neuronal migration. The identification of many causative genes has increased the understanding of normal brain development. A consanguineous family was ascertained with three siblings affected by a severe prenatal neurodevelopmental disorder characterised by fronto-parietal pachygyria, agenesis of the corpus callosum and progressive severe microcephaly. Autozygosity mapping and exome sequencing identified a homozygous novel single base pair deletion, c.1197delT in DMRTA2, predicted to result in a frameshift variant p.(Pro400Leufs*33). DMRTA2 encodes doublesex and mab-3-related transcription factor a2, a transcription factor key to the development of the dorsal telencephalon. Data from murine and zebrafish knockout models are consistent with the variant of DMTRA2 (DMRT5) as responsible for the cortical brain phenotype. Our study suggests that loss of function of DMRTA2 leads to a novel disorder of cortical development.

Understanding the genomic basis of inherited respiratory disorders can assist in the clinical management of individuals with these rare disorders. We apply whole genome sequencing for the discovery of disease-causing variants in the non-coding regions of known disease genes for two individuals with inherited respiratory disorders. We describe analysis strategies to pinpoint candidate non-coding variants within the non-coding genome and demonstrate aberrant RNA splicing as a result of deep intronic variants in DNAH11 and CFTR. These findings confirm clinical diagnoses of primary ciliary dyskinesia and cystic fibrosis, respectively.

BackgroundPathogenic variants in BRCA1 or BRCA2 are identified in ~20% of families with multiple individuals with early-onset breast/ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1/2 have not explained the missing heritability. For the first time, we report transgenerational epigenetic silencing of BRCA1 due to promoter hypermethylation in two families with breast/ovarian cancer.MethodsBRCA1 promoter methylation of ten CpG dinucleotides in breast/ovarian cancer families without germline BRCA1/2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants.FindingsBRCA1 promoter hypermethylation was identified in two of 49 families with multiple women affected with grade 3 breast/high grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa and hair follicles. Methylation levels were ~50%, consistent with the silencing of one allele and confirmed by clonal bisulfite sequencing. RNA sequencing revealed allelic loss of BRCA1 expression in both families and this segregated with a novel heterozygous variant c.-107A>T in the BRCA1 5’UTR.InterpretationOur results indicate a novel mechanism for familial breast/ovarian cancer, caused by epigenetic silencing of the BRCA1 promoter, segregating with an in cis 5’UTR variant in two independent families. We propose that methylation analyses are indicated in all families affected by early onset breast/ovarian cancer without a BRCA1/2 pathogenic variant.FundingFunded by Prevent Breast Cancer (GA 12-006 and GA 15-002) and the Manchester NIHR Biomedical Research Centre (IS-BRC-1215-20007).

Previously genome-wide association methods in patients with classic bladder exstrophy (CBE) found association with ISL1, a master control gene expressed in pericloacal mesenchyme. This study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. Genotypes of 12 markers obtained from 268 CBE patients of Australian, British, German Italian, Spanish and Swedish origin and 1,354 ethnically matched controls and from 92 CBE case-parent trios from North America were analysed. Only marker rs6874700 at the ISL1 locus showed association (p = 2.22 × 10−08). A meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10−19. Developmental biology models were used to clarify the location of ISL1 activity in the forming urinary tract. Genetic lineage analysis of Isl1-expressing cells by the lineage tracer mouse model showed Isl1-expressing cells in the urinary tract of mouse embryos at E10.5 and distributed in the bladder at E15.5. Expression of isl1 in zebrafish larvae staged 48 hpf was detected in a small region of the developing pronephros. Our study supports ISL1 as a major susceptibility gene for CBE and as a regulator of urinary tract development.

Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia. This study investigated the level of HSPA gene expression in human cell lines exposed to hypoxic conditions. Three human glioma cell lines were selected for this study, each representing different types of glioma (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in three glioma cell lines were approximately sixfold higher than in a normal astrocyte cell line. The expression of HSPA was induced in normal cell lines immediately after exposure to hypoxia with 33% of cells exhibiting expression. However, the effects of hypoxia on gene expression were marginal in glioma cells, due to the already increased levels of HSPA with both pre- and post-hypoxia samples showing expression in approximately 90% of cells. These results show that whilst the stress caused by both cancer and hypoxia induce HSPA expression the underlying imprint of tumourgenesis leads to sustained expression.

Melkersson Rosenthal syndromes (MRS) is a rare autosomal dominantly inherited neurocutaneous syndrome characterised by a triad of facial (seventh cranial) nerve palsy, recurrent orofacial swelling and fissuring of the tongue. A recent report implicated a heterozygous missense variant in SLC27A1 (FATP1) as the cause of this condition in members of an affected Chinese family. We undertook Sanger sequencing of this gene in 14 affected unrelated individuals affected by MRS. We did not detect any putative pathogenic variants. Our data indicates that there is both clinical and genetic heterogeneity in this condition and that the causative gene remains to be identified for the majority of cases.