Functional analysis of germline VANGL2 variants using rescue assays of vangl2 knockout zebrafish.

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.

Inactivation of DRG1, encoding a translation factor GTPase, causes a recessive neurodevelopmental disorder.

PURPOSE: Developmentally regulated Guanosine-5'-triphosphate-binding protein 1 (DRG1) is a highly conserved member of a class of GTPases implicated in translation. Although the expression of mammalian DRG1 is elevated in the central nervous system during development, and its function has been implicated in fundamental cellular processes, no pathogenic germline variants have yet been identified. Here, we characterize the clinical and biochemical consequences of DRG1 variants. METHODS: We collate clinical information of 4 individuals with germline DRG1 variants and use in silico, in vitro, and cell-based studies to study the pathogenicity of these alleles. RESULTS: We identified private germline DRG1 variants, including 3 stop-gained p.Gly54∗, p.Arg140∗, p.Lys263∗, and a p.Asn248Phe missense variant. These alleles are recessively inherited in 4 affected individuals from 3 distinct families and cause a neurodevelopmental disorder with global developmental delay, primary microcephaly, short stature, and craniofacial anomalies. We show that these loss-of-function variants (1) severely disrupt DRG1 messenger RNA/protein stability in patient-derived fibroblasts, (2) impair its GTPase activity, and (3) compromise its binding to partner protein ZC3H15. Consistent with the importance of DRG1 in humans, targeted inactivation of mouse Drg1 resulted in preweaning lethality. CONCLUSION: Our work defines a new Mendelian disorder of DRG1 deficiency. This study highlights DRG1's importance for normal mammalian development and underscores the significance of translation factor GTPases in human physiology and homeostasis.

Multicenter study on the genetics of glomerular diseases among southeast and south Asians: Deciphering Diversities - Renal Asian Genetics Network (DRAGoN).

Multinational studies have reported monogenic etiologies in 25%-30% of children with steroid-resistant nephrotic syndrome. Such large studies are lacking in Asia. We established Deciphering Diversities: Renal Asian Genetics Network (DRAGoN) and aimed to describe the genetic and clinical spectrums in Asians. We prospectively studied a cohort of 183 probands with suspected genetic glomerulopathies from South and Southeast Asia, of whom 17% had positive family history. Using multi-gene panel sequencing, we detected pathogenic variants in 26 (14%) probands, of whom one-third had COL4A4 or COL4A5 variants (n = 9, 5%). Of those with COL4A5 defects, only 25% had features suggestive of Alport syndrome. Besides traditional predictors for genetic disease (positive family history and extrarenal malformations), we identified novel predictors, namely older age (6.2 vs. 2.4 years; p = 0.001), hematuria (OR 5.6; 95% CI 2.1-14.8; p < 0.001), and proteinuria in the absence of nephrotic syndrome (OR 4.6; 95% CI 1.8-11.8; p = 0.001) at first manifestation. Among patients who first presented with proteinuria without nephrotic syndrome, the genetic diagnostic rates were >60% when a second risk factor (positive family history or extrarenal manifestation) co-existed. The genetic spectrum of glomerulopathies appears different in Asia. Collagen IV genes may be included in sequencing panels even when suggestive clinical features are absent.

Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell Myelination, Are Responsible for Arthrogryposis Multiplex Congenita.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Heterozygous missense variant in EIF6 gene: A novel form of Shwachman-Diamond syndrome?

Shwachman-Diamond syndrome (SDS) is a rare multisystem ribosomal biogenesis disorder characterized by exocrine pancreatic insufficiency, hematologic abnormalities and bony abnormalities. About 90% of patients have biallelic mutations in SBDS gene. Three additional genes-EFL1, DNAJC21 and SRP54 have been reported in association with a SDS phenotype. However, the cause remains unknown for ~10% of patients. Herein, we report a 6-year-old Chinese boy, who presented in the neonatal period with pancytopenia, liver transaminitis with hepatosplenomegaly and developmental delay, and subsequently developed pancreatic insufficiency complicated by malabsorption and poor growth. Exome sequencing identified a novel de novo heterozygous variant in EIF6 (c.182G>T, p.Arg61Leu). EIF6 protein inhibits ribosomal maturation and is removed in the late steps of ribosomal maturation by SBDS and EFL1 protein. Given the interaction of EIF6 with SBDS and EFL1, we postulate heterozygous variants in EIF6 as a novel cause of Shwachman-Diamond-like phenotype. We compared the phenotype of our patient with those in patients with mutation in SBDS, EFL1, DNAJC21, and SRP54 genes to support this association. Identification of more cases of this novel phenotype would strengthen the association with the genetic etiology.

Cenani-Lenz syndactyly syndrome - a case report of a family with isolated syndactyly.

BACKGROUND: Cenani-Lenz Syndactyly (CLS) syndrome is a rare autosomal recessive disorder characterized by syndactyly and oligodactyly of fingers and toes, disorganization and fusion of metacarpals, metatarsals and phalanges, radioulnar synostosis and mesomelic shortness of the limbs, with lower limbs usually being much less affected than upper limbs. CASE PRESENTATION: we report here two patients, born to consanguineous Sri Lankan parents, present with bilateral postaxial oligodactyly limited to upper limbs. While the proband has no noticeable facial dysmorphism, renal impairments or cognitive impairments, his affected sister displays a few mild facial dysmorphic features. Whole exome sequencing of the proband showed a novel deleterious homozygous mutation (c.1348A > G) in the LRP4 gene, resulting in an Ile450-to-Val (I450V) substitution. CONCLUSION: This recessive mutation in LRP4 confirmed the diagnosis of CLS syndrome in two patients present with isolated hand syndactyly. This is the first reported case of CLS syndrome in a family of Sri Lankan origin.

A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

PURPOSE: DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. EXPERIMENTAL DESIGN: We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. RESULTS: Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. CONCLUSIONS: The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Variant landscape of the RYR1 gene based on whole genome sequencing of the Singaporean population.

The RYR1 gene codes for a ryanodine receptor which is a calcium release channel in the skeletal muscle sarcoplasmic reticulum. It is associated with Malignant Hyperthermia (MH) and congenital myopathies including Central Core Disease (CCD), Multiminicore Disease (MMD) and Congenital Fibre-Type Disproportion (CFTD). There is currently little information on the epidemiology of RYR1 variants in Asians. Our study aims to describe the RYR1 variant landscape in a Singapore cohort unselected for RYR1-associated conditions. Data was retrieved from the SG10K pilot project, where whole genome sequencing was performed on volunteers unselected and undetermined for RYR1-associated conditions. Variants were classified based on pathogenicity using databases ClinVar and InterVar. Allele frequencies of pathogenic variants were compared between Chinese, Indians and Malays. Using databases ExAC, GnomAD and GenomeAsia 100k study, we further compared local allele frequencies to those in Europe, America and Asia. Data was analysed using R Commander. Significant P value was set at p < 0.05. Majority of the RYR1 variants were missense mutations. We identified four pathogenic and four likely pathogenic RYR1 variants. All were related to the aforementioned RYR1-associated conditions. There were 6 carriers of RYR1 pathogenic variants amongst 4810 individuals, corresponding to an allele frequency of 0.06%. The prevalence of pathogenic variants was the highest amongst Indians (4 in 1127 individuals) (p = 0.030). Majority of pathogenic and likely pathogenic mutations were missense and located in mutational hotspots. These variants also occurred at higher frequencies in Asians than globally. This study describes the variant landscape of the RYR1 gene in Singapore. This knowledge will facilitate genetic screening for RYR1-related conditions.

Ermin deficiency leads to compromised myelin, inflammatory milieu, and susceptibility to demyelinating insult.

Ermin is an actin-binding protein found almost exclusively in the central nervous system (CNS) as a component of myelin sheaths. Although Ermin has been predicted to play a role in the formation and stability of myelin sheaths, this has not been directly examined in vivo. Here, we show that Ermin is essential for myelin sheath integrity and normal saltatory conduction. Loss of Ermin in mice caused de-compacted and fragmented myelin sheaths and led to slower conduction along with progressive neurological deficits. RNA sequencing of the corpus callosum, the largest white matter structure in the CNS, pointed to inflammatory activation in aged Ermin-deficient mice, which was corroborated by increased levels of microgliosis and astrogliosis. The inflammatory milieu and myelin abnormalities were further associated with increased susceptibility to immune-mediated demyelination insult in Ermin knockout mice. Supporting a possible role of Ermin deficiency in inflammatory white matter disorders, a rare inactivating mutation in the ERMN gene was identified in multiple sclerosis patients. Our findings demonstrate a critical role for Ermin in maintaining myelin integrity. Given its near-exclusive expression in myelinating oligodendrocytes, Ermin deficiency represents a compelling "inside-out" model of inflammatory dysmyelination and may offer a new paradigm for the development of myelin stability-targeted therapies.

Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

A loss-of-function NUAK2 mutation in humans causes anencephaly due to impaired Hippo-YAP signaling.

Failure of neural tube closure during embryonic development can result in anencephaly, one of the most common birth defects in humans. A family with recurrent anencephalic fetuses was investigated to understand its etiology and pathogenesis. Exome sequencing revealed a recessive germline 21-bp in-frame deletion in NUAK2 segregating with the disease. In vitro kinase assays demonstrated that the 7-amino acid truncation in NUAK2, a serine/threonine kinase, completely abrogated its catalytic activity. Patient-derived disease models including neural progenitor cells and cerebral organoids showed that loss of NUAK2 activity led to decreased Hippo signaling via cytoplasmic YAP retention. In neural tube-like structures, endogenous NUAK2 colocalized apically with the actomyosin network, which was disrupted in patient cells, causing impaired nucleokinesis and apical constriction. Our results establish NUAK2 as an indispensable kinase for brain development in humans and suggest that a NUAK2-Hippo signaling axis regulates cytoskeletal processes that govern cell shape during neural tube closure.

Bone matrix hypermineralization associated with low bone turnover in a case of Nasu-Hakola disease.

Analysis of tissue from a 34-years-old male patient from Austrian origin with a history of multiple fractures associated with painful episodes over the carpal, tarsal and at the end of the long bones respectively is presented. Radiographic images and axial 3DCT scans showed widespread defects in trabecular bone architecture and ill-defined cortices over these skeletal sites in the form of discrete cystic-like lesions. Family history indicated two sisters (one half and one full biological sisters) also with a history of fractures. Whole exome sequencing revealed two heterozygous missense mutations in TYROBP (MIM 604142; NM_003332.3) gene encoding for a cell-surface adaptor protein, which is part of a signaling complex triggering activation of immune responses. It is expressed in cells of the ectoderm cell linage such as NK and dendritic cells, macrophages, monocytes, myeloid cells, microglia cells and osteoclasts. The phenotype and genotype of the patient were consistent with the diagnosis of Nasu-Hakola disease (NHD) (OMIM 221770). Investigations at the bone material level of a transiliac bone biopsy sample from the patient using polarized light microscopy and backscatter electron imaging revealed disordered lamellar collagen fibril arrangement and extensively increased matrix mineralization. These findings are the first bone material data in a patient with NHD and point toward an osteoclast defect involvement in this genetic condition.

Novel mutations in the ciliopathy-associated gene CPLANE1 (C5orf42) cause OFD syndrome type VI rather than Joubert syndrome.

Mutations in CPLANE1 (previously known as C5orf42) cause Oral-Facial-Digital Syndrome type VI (OFD6) as well as milder Joubert syndrome (JS) phenotypes. Seven new cases from five unrelated families diagnosed with pure OFD6 were systematically examined. Based on the clinical manifestations of these patients and those described in the literature, we revised the diagnostic features of OFD6 and include the seven most common characteristics: 1) molar tooth sign, 2) tongue hamartoma and/or lobulated tongue, 3) additional frenula, 4) mesoaxial polydactyly of hands, 5) preaxial polydactyly of feet, 6) syndactyly and/or bifid toe, and 7) hypothalamic hamartoma. By whole or targeted exome sequencing, we identified seven novel germline recessive mutations in CPLANE1, including missense, nonsense, frameshift and canonical splice site variants, all causing OFD6 in these patients. Since CPLANE1 is also mutated in JS patients, we examined whether a genotype-phenotype correlation could be established. We gathered and compared 46 biallelic CPLANE1 mutations reported in 32 JS and 26 OFD6 patients. Since no clear correlation between paired genotypes and clinical outcomes could be determined, we concluded that patient's genetic background and gene modifiers may modify the penetrance and expressivity of CPLANE1 causal alleles. To conclude, our study provides a comprehensive view of the phenotypic range, the genetic basis and genotype-phenotype association in OFD6 and JS. The updated phenotype scoring system together with the identification of new CPLANE1 mutations will help clinicians and geneticists reach a more accurate diagnosis for JS-related disorders.

Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells.

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.

A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome.

BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. RESULTS: Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. CONCLUSIONS: Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.

Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network.

The bacterial pathogen Xanthomonas campestris pv. campestris co-ordinates virulence factor production and biofilm dispersal through a diffusible signal factor (DSF)-mediated cell-cell communication mechanism. The RpfC/RpfG two-component system plays a key role in DSF signal transduction and appears to modulate downstream DSF regulon by changing intracellular content of cyclic dimeric GMP (c-di-GMP), an unusual nucleotide second messenger. Here we show that Clp, a conserved global regulator showing a strong homology to the cAMP nucleotide receptor protein Crp of Escherichia coli, is essential for DSF regulation of virulence factor production but not for biofilm dispersal. Deletion of clp in Xcc changed the transcriptional expression of 299 genes including a few encoding transcription factors. Further genetic and microarray analysis led to identification of a homologue of the transcriptional regulator Zur, and a novel TetR-type transcription factor FhrR. These two regulatory factors regulated different sets of genes within Clp regulon. These results outline a hierarchical signalling network by which DSF modulates different biological functions, and may also provide a clue on how the novel nucleotide signal can be coupled to its downstream regulatory networks.