De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.

Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

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.

Clinical utility of NGS diagnosis and disease stratification in a multiethnic primary ciliary dyskinesia cohort.

BACKGROUND: Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests. METHODS: The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. RESULTS: Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results. CONCLUSIONS: This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

Tablet and web-based audiometry to screen for hearing loss in adults with cystic fibrosis.

INTRODUCTION: Individuals with chronic lung disease (eg, cystic fibrosis (CF)) often receive antimicrobial therapy including aminoglycosides resulting in ototoxicity. Extended high-frequency audiometry has increased sensitivity for ototoxicity detection, but diagnostic audiometry in a sound-booth is costly, time-consuming and requires a trained audiologist. This cross-sectional study analysed tablet-based audiometry (Shoebox MD) performed by non-audiologists in an outpatient setting, alongside home web-based audiometry (3D Tune-In) to screen for hearing loss in adults with CF. METHODS: Hearing was analysed in 126 CF adults using validated questionnaires, a web self-hearing test (0.5 to 4 kHz), tablet (0.25 to 12 kHz) and sound-booth audiometry (0.25 to 12 kHz). A threshold of ≥25 dB hearing loss at ≥1 audiometric frequency was considered abnormal. Demographics and mitochondrial DNA sequencing were used to analyse risk factors, and accuracy and usability of hearing tests determined. RESULTS: Prevalence of hearing loss within any frequency band tested was 48%. Multivariate analysis showed age (OR 1.127; (95% CI: 1.07 to 1.18; p value<0.0001) per year older) and total intravenous antibiotic days over 10 years (OR 1.006; (95% CI: 1.002 to 1.010; p value=0.004) per further intravenous day) were significantly associated with increased risk of hearing loss. Tablet audiometry had good usability, was 93% sensitive, 88% specific with 94% negative predictive value to screen for hearing loss compared with web self-test audiometry and questionnaires which had poor sensitivity (17% and 13%, respectively). Intraclass correlation (ICC) of tablet versus sound-booth audiometry showed high correlation (ICC >0.9) at all frequencies ≥4 kHz. CONCLUSIONS: Adults with CF have a high prevalence of drug-related hearing loss and tablet-based audiometry can be a practical, accurate screening tool within integrated ototoxicity monitoring programmes for early detection.

Classification and correlation of RYR2 missense variants in individuals with catecholaminergic polymorphic ventricular tachycardia reveals phenotypic relationships.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is predominantly caused by heterozygous missense variants in the cardiac ryanodine receptor, RYR2. However, many RYR2 missense variants are classified as variants of uncertain significance (VUS). We systematically re-evaluated all RYR2 variants in healthy individuals and those with CPVT or arrhythmia using the 2015 American College of Medical Genomics guidelines. RYR2 variants were identified by the NW Genomic Laboratory Hub, from the published literature and databases of sequence variants. Each variant was assessed based on minor allele frequencies, in silico prediction tools and appraisal of functional studies and classified according to the ACMG-AMP guidelines. Phenotype data was collated where available. Of the 326 identified RYR2 missense variants, 55 (16.9%), previously disease-associated variants were reclassified as benign. Application of the gnomAD database of >140,000 controls allowed reclassification of 11 variants more than the ExAC database. CPVT-associated RYR2 variants clustered predominantly between amino acid positions 3949-4332 and 4867-4967 as well as the RyR and IP3R homology-associated and ion transport domains (p < 0.005). CPVT-associated RYR2 variants occurred at more conserved amino acid positions compared with controls, and variants associated with sudden death had higher conservation scores (p < 0.005). There were five potentially pathogenic RYR2 variants associated with sudden death during sleep which were located almost exclusively in the C-terminus of the protein. In conclusion, control sequence databases facilitate reclassification of RYR2 variants but the majority remain as VUS. Notably, pathogenic variants in RYR2 are associated with death in sleep.

BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.

CardioClassifier: disease- and gene-specific computational decision support for clinical genome interpretation.

PURPOSE: Internationally adopted variant interpretation guidelines from the American College of Medical Genetics and Genomics (ACMG) are generic and require disease-specific refinement. Here we developed CardioClassifier ( http://www.cardioclassifier.org ), a semiautomated decision-support tool for inherited cardiac conditions (ICCs). METHODS: CardioClassifier integrates data retrieved from multiple sources with user-input case-specific information, through an interactive interface, to support variant interpretation. Combining disease- and gene-specific knowledge with variant observations in large cohorts of cases and controls, we refined 14 computational ACMG criteria and created three ICC-specific rules. RESULTS: We benchmarked CardioClassifier on 57 expertly curated variants and show full retrieval of all computational data, concordantly activating 87.3% of rules. A generic annotation tool identified fewer than half as many clinically actionable variants (64/219 vs. 156/219, Fisher's P = 1.1 × 10-18), with important false positives, illustrating the critical importance of disease and gene-specific annotations. CardioClassifier identified putatively disease-causing variants in 33.7% of 327 cardiomyopathy cases, comparable with leading ICC laboratories. Through addition of manually curated data, variants found in over 40% of cardiomyopathy cases are fully annotated, without requiring additional user-input data. CONCLUSION: CardioClassifier is an ICC-specific decision-support tool that integrates expertly curated computational annotations with case-specific data to generate fast, reproducible, and interactive variant pathogenicity reports, according to best practice guidelines.

Autosomal Recessive Primary Microcephaly (MCPH): An Update.

Autosomal recessive primary microcephaly (MCPH; MicroCephaly Primary Hereditary) is a genetically heterogeneous neurodevelopmental disorder characterized by a significantly reduced head circumference present already at birth and intellectual disability. Inconsistent features include hyperactivity, an expressive speech disorder, and epilepsy. Here, we provide a brief overview on this rare disorder pertinent for clinicians.

Transplantation from a symptomatic carrier sister restores host defenses but does not prevent colitis in NEMO deficiency.

NF-κB essential modulator (NEMO) deficiency causes ectodermal dysplasia with immunodeficiency in males, while manifesting as incontinentia pigmenti in heterozygous females. We report a family with NEMO deficiency, in which a female carrier displayed skewed X-inactivation favoring the mutant NEMO allele associated with symptoms of Behçet's disease. Hematopoietic stem cell transplantation of an affected boy from this donor reconstituted an immune system with retained skewed X-inactivation. After transplantation no more severe infections occurred, indicating that an active wild-type NEMO allele in only 10% of immune cells restores host defense. Yet he developed inflammatory bowel disease (IBD). While gut infiltrating immune cells stained strongly for nuclear p65 indicating restored NEMO function, this was not the case in intestinal epithelial cells - in contrast to cells from conventional IBD patients. These results extend murine observations that epithelial NEMO-deficiency suffices to cause IBD. High anti-TNF doses controlled the intestinal inflammation and symptoms of Behçet's disease.

Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans.

blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.

Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function in the V2 vasopressin receptor (V2R). These receptor mutations lead to hyponatremia, which may lead to clinical symptoms in infants. Here we present a newly identified I130N substitution in exon 2 of the V2R gene in a family, causing NSIAD. This I130N mutation resulted in constitutive activity of the V2R with constitutive cyclic adenosine monophosphate (cAMP) generation in HEK293 cells. This basal activity could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation as the basal cAMP generation activity of I130N does not lead to interaction with ß-arrestin. The constitutive activity of the mutant receptor caused constitutive dynamin-dependent and ß-arrestin-independent internalization. The inhibition of basal internalization using dominant-negative dynamin resulted in an increased cell surface expression. In contrast to the constitutive internalization, agonist-induced endocytosis was ß-arrestin dependent. Thus, tolvaptan could be used for treatment of hyponatremia in patients with NSIAD who carry the I130N-V2R mutation.

STIL mutation causes autosomal recessive microcephalic lobar holoprosencephaly.

Holoprosencephaly is a clinically and genetically heterogeneous midline brain malformation associated with neurologic manifestations including developmental delay, intellectual disability and seizures. Although mutations in the sonic hedgehog gene SHH and more than 10 other genes are known to cause holoprosencephaly, many patients remain without a molecular diagnosis. Here we show that a homozygous truncating mutation of STIL not only causes severe autosomal recessive microcephaly, but also lobar holoprosencephaly in an extended consanguineous Pakistani family. STIL mutations have previously been linked to centrosomal defects in primary microcephaly at the MCPH7 locus. Our results thus expand the clinical phenotypes associated with biallellic STIL mutations to include holoprosencephaly.

What next-generation sequencing (NGS) technology has enabled us to learn about primary autosomal recessive microcephaly (MCPH).

The impact that next-generation sequencing technology (NGS) is having on many aspects of molecular and cell biology, is becoming increasingly apparent. One of the most noticeable outcomes of the new technology in human genetics, has been the accelerated rate of identification of disease-causing genes. Especially for rare, heterogeneous disorders, such as autosomal recessive primary microcephaly (MCPH), the handful of genes previously known to harbour disease-causing mutations, has grown at an unprecedented rate within a few years. Knowledge of new genes mutated in MCPH over the last four years has contributed to our understanding of the disorder at both the clinical and cellular levels. The functions of proteins such as WDR62, CASC5, PHC1, CDK6, CENP-E, CENP-F, CEP63, ZNF335, PLK4 and TUBGPC, have been added to the complex network of critical cellular processes known to be involved in brain growth and size. In addition to the importance of mitotic spindle assembly and structure, centrosome and centriole function and DNA repair and damage response, new mechanisms involving kinetochore-associated proteins and chromatin remodelling complexes have been elucidated. Two of the major contributions to our clinical knowledge are the realisation that primary microcephaly caused by mutations in genes at the MCPH loci is seldom an isolated clinical feature and is often accompanied either by additional cortical malformations or primordial dwarfism. Gene-phenotype correlations are being revisited, with a new dimension of locus heterogeneity and phenotypic variability being revealed.

Severe presentation of WDR62 mutation: is there a role for modifying genetic factors?

Mutations in WDR62 are associated with primary microcephaly; however, they have been reported with wide phenotypic variability. We report on six individuals with novel WDR62 mutations who illustrate this variability and describe three in greater detail. Of the three, one lacks neuromotor development and has severe pachygyria on MRI, another has only delayed speech and motor development and moderate polymicrogyria, and the third has an intermediate phenotype. We observed a rare copy number change of unknown significance, a 17q25qter duplication, in the first severely affected individual. The 17q25 duplication included an interesting candidate gene, tubulin cofactor D (TBCD), crucial in microtubule assembly and disassembly. Sequencing of the non-duplicated allele showed a TBCD missense mutation, predicted to cause a deleterious p.Phe1121Val substitution. Sequencing of a cohort of five patients with WDR62 mutations, including one with an identical mutation and different phenotype, plus 12 individuals with diagnosis of microlissencephaly and another individual with mild intellectual disability (ID) and a 17q25 duplication, did not reveal TBCD mutations. However, immunostaining with tubulin antibodies of cells from patients with both WDR62 and TBCD mutation showed abnormal tubulin network when compared to controls and cells with only the WDR62 mutation. Therefore, we propose that genetic factors contribute to modify the severity of the WDR62 phenotype and, although based on suggestive evidence, TBCD could function as one of such factors.

Lethal course of meconium ileus in preterm twins revealing a novel cystic fibrosis mutation (p.Cys524Tyr).

BACKGROUND: In term newborns meconium ileus is frequently associated with cystic fibrosis. Reports on meconium ileus in preterm infants being diagnosed with cystic fibrosis early after birth are very scarce. Associations between genotype and phenotype in cystic fibrosis and its particular comorbidities have been reported. CASE PRESENTATION: Two extremely preterm twin infants (26 weeks of gestation) born from a Malaysian mother and a Caucasian father were presented with typical signs of meconium ileus. Despite immediate surgery both displayed a unique and finally lethal course. Mutation analysis revealed a novel, probably pathogenic cystic fibrosis mutation, p.Cys524Tyr. The novel mutation might explain the severity of disease next to typical sequelae of prematurity. CONCLUSION: Preterm neonates with meconium ileus have to be evaluated for cystic fibrosis beyond ethnical boundaries, but may take devastating clinical courses despite early treatment. The novel, potentially pathogenic CF mutation p.Cys524Tyr might be associated with severe meconium ileus in neonates. Disease-modifying loci are important targets for intestinal comorbidity of cystic fibrosis.

Combined approaches, including long-read sequencing, address the diagnostic challenge of HYDIN in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD), a disorder of the motile cilia, is now recognised as an underdiagnosed cause of bronchiectasis. Accurate PCD diagnosis comprises clinical assessment, analysis of cilia and the identification of biallelic variants in one of 50 known PCD-related genes, including HYDIN. HYDIN-related PCD is underdiagnosed due to the presence of a pseudogene, HYDIN2, with 98% sequence homology to HYDIN. This presents a significant challenge for Short-Read Next Generation Sequencing (SR-NGS) and analysis, and many diagnostic PCD gene panels do not include HYDIN. We have used a combined approach of SR-NGS with bioinformatic masking of HYDIN2, and state-of-the-art long-read Nanopore sequencing (LR_NGS), together with analysis of respiratory cilia including transmission electron microscopy and immunofluorescence to address the underdiagnosis of HYDIN as a cause of PCD. Bioinformatic masking of HYDIN2 after SR-NGS facilitated the detection of biallelic HYDIN variants in 15 of 437 families, but compromised the detection of copy number variants. Supplementing testing with LR-NGS detected HYDIN deletions in 2 families, where SR-NGS had detected a single heterozygous HYDIN variant. LR-NGS was also able to confirm true homozygosity in 2 families when parental testing was not possible. Utilising a combined genomic diagnostic approach, biallelic HYDIN variants were detected in 17 families from 242 genetically confirmed PCD cases, comprising 7% of our PCD cohort. This represents the largest reported HYDIN cohort to date and highlights previous underdiagnosis of HYDIN-associated PCD. Moreover this provides further evidence for the utility of LR-NGS in diagnostic testing, particularly for regions of high genomic complexity.