A gene pathogenicity tool "GenePy" identifies missed biallelic diagnoses in the 100,000 Genomes Project.

PURPOSE: The 100,000 Genomes Project diagnosed a quarter of affected participants, but 26% of diagnoses were not on the applied gene panel(s); with many being de novo variants. Assessing biallelic variants without a gene panel is more challenging. METHODS: We sought to identify missed biallelic diagnoses using GenePy, which incorporates allele frequency, zygosity, and a user-defined deleterious metric, generating an aggregate GenePy score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 100,000 Genomes Project participants. For each gene, we ranked participant GenePy scores and scrutinized affected participants without a diagnosis, whose scores ranked among the top 5 for each gene. In cases which participant phenotypes overlapped with the disease gene of interest, we extracted rare variants and applied phase, ClinVar, and ACMG classification. RESULTS: 3184 affected individuals without a molecular diagnosis had a top-5-ranked GenePy score and 682 of 3184 (21%) had phenotypes overlapping with a top-ranking gene. In 122 of 669 (18%) phenotype-matched cases (excluding 13 withdrawn participants), we identified a putative missed diagnosis (2.2% of all undiagnosed participants). A further 334 of 669 (50%) cases have a possible missed diagnosis but require functional validation. CONCLUSION: Applying GenePy at scale has identified 456 potential diagnoses, demonstrating the value of novel diagnostic strategies.

A Panel-Agnostic Strategy 'HiPPo' Improves Diagnostic Efficiency in the UK Genomic Medicine Service.

Genome sequencing is available as a clinical test in the UK through the Genomic Medicine Service (GMS). The GMS analytical strategy predominantly filters genome data on preselected gene panels. Whilst this reduces variants requiring assessment by reporting laboratories, pathogenic variants outside applied panels may be missed, and variants in genes without established disease-gene relationships are largely ignored. This study compares the analysis of a research exome to a GMS clinical genome for the same patients. For the research exome, we applied a panel-agnostic approach filtering for variants with High Pathogenic Potential (HiPPo) using ClinVar, allele frequency, and in silico prediction tools. We then restricted HiPPo variants to Gene Curation Coalition (GenCC) disease genes. These results were compared with the GMS genome panel-based approach. Twenty-four participants from eight families underwent parallel research exome and GMS genome sequencing. Exome HiPPo analysis identified a similar number of variants as the GMS panel-based approach. GMS genome analysis returned two pathogenic variants and one de novo variant. Exome HiPPo analysis returned the same variants plus an additional pathogenic variant and three further de novo variants in novel genes, where case series are underway. When HiPPo was restricted to GenCC disease genes, statistically fewer variants required assessment to identify more pathogenic variants than reported by the GMS, giving a diagnostic rate per variant assessed of 20% for HiPPo versus 3% for the GMS. With UK plans to sequence 5 million genomes, strategies are needed to optimise genome analysis beyond gene panels whilst minimising the burden of variants requiring clinical assessment.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project.

BACKGROUND: Current clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm of the 100 000 Genomes Project (100kGP), families were recruited under the clinical indication 'single autosomal recessive mutation in rare disease'. These participants presented with strong clinical suspicion for a specific autosomal recessive disorder, but only one suspected pathogenic variant had been identified through standard-of-care testing. Whole genome sequencing (WGS) aimed to identify cryptic 'second-hit' variants. METHODS: To investigate the 31 families with available data that remained unsolved following formal review within the 100kGP, SVRare was used to aggregate structural variants present in <1% of 100kGP participants. Small variants were assessed using population allele frequency data and SpliceAI. Literature searches and publicly available online tools were used for further annotation of pathogenicity. RESULTS: Using these strategies, 8/31 cases were solved, increasing the overall diagnostic yield of this cohort from 10/41 (24.4%) to 18/41 (43.9%). Exemplar cases include a patient with cystic fibrosis harbouring a novel exonic LINE1 insertion in CFTR and a patient with generalised arterial calcification of infancy with complex interlinked duplications involving exons 2-6 of ENPP1. Although ambiguous by short-read WGS, the ENPP1 variant structure was resolved using optical genome mapping and RNA analysis. CONCLUSION: Systematic examination of cryptic variants across a multi-disease cohort successfully identifies additional pathogenic variants. WGS data analysis in autosomal recessive rare disease should consider complex structural and small intronic variants as potentially pathogenic second hits.

A gene pathogenicity tool 'GenePy' identifies missed biallelic diagnoses in the 100,000 Genomes Project.

The 100,000 Genomes Project (100KGP) diagnosed a quarter of recruited affected participants, but 26% of diagnoses were in genes not on the chosen gene panel(s); with many being de novo variants of high impact. However, assessing biallelic variants without a gene panel is challenging, due to the number of variants requiring scrutiny. We sought to identify potential missed biallelic diagnoses independent of the gene panel applied using GenePy - a whole gene pathogenicity metric. GenePy scores all variants called in a given individual, incorporating allele frequency, zygosity, and a user-defined deleterious metric (CADD v1.6 applied herein). GenePy then combines all variant scores for individual genes, generating an aggregate score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 individuals in 100KGP. For each gene, we ranked participant GenePy scores for that gene, and scrutinised affected individuals without a diagnosis whose scores ranked amongst the top-5 for each gene. We assessed these participants' phenotypes for overlap with the disease gene associated phenotype for which they were highly ranked. Where phenotypes overlapped, we extracted rare variants in the gene of interest and applied phase, ClinVar and ACMG classification looking for putative causal biallelic variants. 3184 affected individuals without a molecular diagnosis had a top-5 ranked GenePy gene score and 682/3184 (21%) had phenotypes overlapping with one of the top-ranking genes. After removing 13 withdrawn participants, in 122/669 (18%) of the phenotype-matched cases, we identified a putative missed diagnosis in a top-ranked gene supported by phasing, ClinVar and ACMG classification. A further 334/669 (50%) of cases have a possible missed diagnosis but require functional validation. Applying GenePy at scale has identified potential diagnoses for 456/3183 (14%) of undiagnosed participants who had a top-5 ranked GenePy score in a recessive disease gene, whilst adding only 1.2 additional variants (per individual) for assessment.

The Palestinian primary ciliary dyskinesia population: first results of the diagnostic and genetic spectrum.

Background: Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population. Methods: Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV1) Global Lung Index z-scores and body mass index z-scores. Results: 68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes. CCDC39, DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median -1.90 (-5.0-1.32)) and growth was mostly within the normal range (z-score mean -0.36 (-3.03-2.57). 19% individuals had finger clubbing. Conclusions: Despite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity.

A panel-agnostic strategy 'HiPPo' improves diagnostic efficiency in the UK Genome Medicine Service.

Genome sequencing is now available as a clinical test on the National Health Service (NHS) through the Genome Medicine Service (GMS). The GMS have set out an analytical strategy that predominantly filters genome data on a pre-selected gene panel(s). Whilst this approach reduces the number of variants requiring assessment by reporting laboratories, pathogenic variants outside of the gene panel applied may be missed, and candidate variants in novel genes are largely ignored. This study sought to compare a research exome analysis to an independent clinical genome analysis performed through the NHS for the same group of patients. When analysing the exome data, we applied a panel agnostic approach filtering for variants with High Pathogenic Potential (HiPPo) using ClinVar, allele frequency, and in silico prediction tools. We then compared this gene agnostic analysis to the panel-based approach as applied by the GMS to genome data. Later we restricted HiPPo variants to a panel of the Gene Curation Coalition (GenCC) morbid genes and compared the diagnostic yield with the variants filtered using the GMS strategy. 24 patients from 8 families underwent parallel research exome sequencing and GMS genome sequencing. HiPPo analysis applied to research exome data identified a similar number of variants as the gene panel-based approach applied by the GMS. GMS clinical genome analysis identified and returned 2 pathogenic variants and 3 variants of uncertain significance. HiPPo research exome analysis identified the same variants plus an additional pathogenic variant and a further 3 de novo variants of uncertain significance in novel genes, where case series and functional studies are underway. When HiPPo was restricted to GenCC disease genes (strong or definitive), the same pathogenic variants were identified yet statistically fewer variants required assessment to identify more diagnostic variants than reported by the GMS genome strategy. This gave a diagnostic rate per variant assessed of 20% for HiPPo restricted to GenCC versus 3% for the GMS panel-based approach. With plans to sequence 5 million more NHS patients, strategies are needed to optimise the full potential of genome data beyond gene panels whilst minimising the burden of variants that require clinical assessment.

Biallelic variants in CEP164 cause a motile ciliopathy-like syndrome.

Ciliopathies may be classed as primary or motile depending on the underlying ciliary defect and are usually considered distinct clinical entities. Primary ciliopathies are associated with multisystem syndromes typically affecting the brain, kidney, and eye, as well as other organ systems such as the liver, skeleton, auditory system, and metabolism. Motile ciliopathies are a heterogenous group of disorders with defects in specialised motile ciliated tissues found within the lung, brain, and reproductive system, and are associated with primary ciliary dyskinesia, bronchiectasis, infertility and rarely hydrocephalus. Primary and motile cilia share defined core ultra-structures with an overlapping proteome, and human disease phenotypes can reflect both primary and motile ciliopathies. CEP164 encodes a centrosomal distal appendage protein vital for primary ciliogenesis. Human CEP164 mutations are typically described in patients with nephronophthisis-related primary ciliopathies but have also been implicated in motile ciliary dysfunction. Here we describe a patient with an atypical motile ciliopathy phenotype and biallelic CEP164 variants. This work provides further evidence that CEP164 mutations can contribute to both primary and motile ciliopathy syndromes, supporting their functional and clinical overlap, and informs the investigation and management of CEP164 ciliopathy patients.

Targeting de novo loss-of-function variants in constrained disease genes improves diagnostic rates in the 100,000 Genomes Project.

BACKGROUND: Genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP). Analysis was restricted to predefined gene panels associated with the patient's phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnoses outside of the panel(s) applied. We propose a complementary method to rapidly identify pathogenic variants, including those missed by 100KGP methods. METHODS: The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, loss-of-function variants in disease genes with a LOEUF score < 0.2. We compared our findings with the corresponding patient's diagnostic reports. RESULTS: 324/332 (98%) of the variants identified using DeNovoLOEUF were diagnostic or partially diagnostic (whereby the variant was responsible for some of the phenotype). We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients. CONCLUSION: We have demonstrated a highly specific and rapid method with a 98% positive predictive value that has good concordance with standard analysis, low false-positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered.

A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project.

BACKGROUND: Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. METHODS: Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon-intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon-intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. RESULTS: We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. CONCLUSIONS: Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases.

Evidence that the Ser192Tyr/Arg402Gln in cis Tyrosinase gene haplotype is a disease-causing allele in oculocutaneous albinism type 1B (OCA1B).

Oculocutaneous albinism type 1 (OCA1) is caused by pathogenic variants in the TYR (tyrosinase) gene which encodes the critical and rate-limiting enzyme in melanin synthesis. It is the most common OCA subtype found in Caucasians, accounting for ~50% of cases worldwide. The apparent 'missing heritability' in OCA is well described, with ~25-30% of clinically diagnosed individuals lacking two clearly pathogenic variants. Here we undertook empowered genetic studies in an extensive multigenerational Amish family, alongside a review of previously published literature, a retrospective analysis of in-house datasets, and tyrosinase activity studies. Together this provides irrefutable evidence of the pathogenicity of two common TYR variants, p.(Ser192Tyr) and p.(Arg402Gln) when inherited in cis alongside a pathogenic TYR variant in trans. We also show that homozygosity for the p.(Ser192Tyr)/p.(Arg402Gln) TYR haplotype results in a very mild, but fully penetrant, albinism phenotype. Together these data underscore the importance of including the TYR p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype as a pathogenic allele causative of OCA, which would likely increase molecular diagnoses in this missing heritability albinism cohort by 25-50%.

Whole genome sequencing in the diagnosis of primary ciliary dyskinesia.

BACKGROUND: It is estimated that 1-13% of cases of bronchiectasis in adults globally are attributable to primary ciliary dyskinesia (PCD) but many adult patients with bronchiectasis have not been investigated for PCD. PCD is a disorder caused by mutations in genes required for motile cilium structure or function, resulting in impaired mucociliary clearance. Symptoms appear in infancy but diagnosis is often late or missed, often due to the lack of a "gold standard" diagnostic tool and non-specific symptoms. Mutations in > 50 genes account for around 70% of cases, with additional genes, and non-coding, synonymous, missense changes or structural variants (SVs) in known genes presumed to account for the missing heritability. METHODS: UK patients with no identified genetic confirmation for the cause of their PCD or bronchiectasis were eligible for whole genome sequencing (WGS) in the Genomics England Ltd 100,000 Genomes Project. 21 PCD probands and 52 non-cystic fibrosis (CF) bronchiectasis probands were recruited in Wessex Genome Medicine Centre (GMC). We carried out analysis of single nucleotide variants (SNVs) and SVs in all families recruited in Wessex GMC. RESULTS: 16/21 probands in the PCD cohort received confirmed (n = 9), probable (n = 4) or possible (n = 3) diagnosis from WGS, although 13/16 of these could have been picked up by current standard of care gene panel testing. In the other cases, SVs were identified which were missed by panel testing. We identified variants in novel PCD candidate genes (IFT140 and PLK4) in 2 probands in the PCD cohort. 3/52 probands in the non-CF bronchiectasis cohort received a confirmed (n = 2) or possible (n = 1) diagnosis of PCD. We identified variants in novel PCD candidate genes (CFAP53 and CEP164) in 2 further probands in the non-CF bronchiectasis cohort. CONCLUSIONS: Genetic testing is an important component of diagnosing PCD, especially in cases of atypical disease history. WGS is effective in cases where prior gene panel testing has found no variants or only heterozygous variants. In these cases it may detect SVs and is a powerful tool for novel gene discovery.

Genetic Analysis of Pediatric Primary Adrenal Insufficiency of Unknown Etiology: 25 Years' Experience in the UK.

CONTEXT: Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood. OBJECTIVE: We investigated genetic causes of PAI in children and young people over a 25 year period. DESIGN SETTING AND PARTICIPANTS: Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers. INTERVENTION AND OUTCOME MEASUREMENTS: Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018). RESULTS: A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1/steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause. CONCLUSIONS: PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved.

Topological data analysis reveals genotype-phenotype relationships in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype-phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases. METHODS: We applied a topological data analysis (TDA) approach to investigate genotype-phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics. RESULTS: Disease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV1) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis. CONCLUSIONS: This large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype-phenotype relationships reported by smaller studies (e.g. FEV1 worse with CCDC39 mutation) and identified new relationships, including FEV1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations.

A CRISPR and high-content imaging assay compliant with ACMG/AMP guidelines for clinical variant interpretation in ciliopathies.

Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31+/- human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.

Screening of a large PAX6 cohort identified many novel variants and emphasises the importance of the paired and homeobox domains.

Pathogenic variants within PAX6 are most often associated with aniridia, but have been linked with other phenotypes such as nystagmus, cataracts and foveal hypoplasia. Data are presented from a large cohort of 434 probands referred for PAX6 diagnostic testing. This analysis identified a wide range of pathogenic variants (n = 145) in 254 probands (including 61 novel variants). Excluding missense variants predicted to affect splicing, all 29 of the remaining missense variants were located within the paired (n = 27) or homeobox (n = 2) domains of the PAX6 protein, providing further evidence that these domains are critical to normal PAX6 function. Genotype-phenotype evidence suggests that while aniridia is associated with most variant types, a much broader clinical spectrum is seen in patients harbouring a missense variant, or a frameshift or run-on variant that results in an elongated or extended PAX6 protein.

Correction: Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

PURPOSE: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. METHODS: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. RESULTS: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity. CONCLUSION: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Screening of a large cohort of blepharophimosis, ptosis, and epicanthus inversus syndrome patients reveals a very strong paternal inheritance bias and a wide spectrum of novel FOXL2 mutations.

Blepharophimosis, Ptosis, and Epicanthus inversus Syndrome (BPES) is caused by autosomal dominant mutations in FOXL2. There are two forms of BPES: type I (with primary ovarian insufficiency (POI)) and type II (without POI). Data are presented from a large cohort of 177 BPES probands. Diagnostic testing identified a wide range of mutations in 119 mutation-positive patients (including 38 novel mutations). Although FOXL2 mutations are distributed throughout the gene, over 50% were frameshift mutations within a hotspot region of the gene that can be detected using a single primer pair to provide a cost-effective and rapid screening method. There was a significant proportion of de novo cases in this study, although in 7% there may be undetected parental mosaicism. There was an excess of female compared to male probands and a highly significant bias in the parental original of inherited mutations, with 20/21 found to be paternal in origin (95%). This could be because BPES in a female is more likely to come to clinical attention and because there is a generalised and more widespread clinical effect on fertility, in addition to the established association with POI. This study demonstrates the importance of cascade screening and provides new information on inheritance and parental mosaicism in BPES which will aid genetic counselling and accurate risk management.

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

DNA methylation profiling in X;autosome translocations supports a role for L1 repeats in the spread of X chromosome inactivation.

X chromosome inactivation (XCI) is an epigenetic mechanism that silences the majority of genes on one X chromosome in females. Previous studies have suggested that the spread of XCI might be facilitated in part by common repeats such as long interspersed nuclear elements (LINEs). However, owing to the unusual sequence content of the X and the nonrandom distribution of genes that escape XCI, it has been unclear whether the correlation between repeat elements and XCI is a functional one. To test the hypothesis that the spread of XCI shows sequence specificity, we have analyzed the pattern of XCI in autosomal chromatin by performing DNA methylation profiling in six unbalanced X;autosome translocations. Using promoter hypermethylation as an epigenetic signature of XCI, we have determined the inactivation status of 1050 autosomal genes after translocation onto an inactive derivative X. By performing a comparative sequence analysis of autosomal genes that are either subject to or escape the X inactivation signal, we identified a number of common repetitive elements, including L1 and L2 LINEs, and DNA motifs that are significantly enriched around inactive autosomal genes. We show that these same motifs predominantly map to L1P repeat elements, are significantly enriched on the X chromosome versus the autosomes and also occur at higher densities around X-linked genes that are subject to X inactivation compared with those that escape X inactivation. These results are consistent with a potential causal relationship between DNA sequence features such as L1s and the spread of XCI, lending strong support to Mary Lyon's 'repeat hypothesis'.