POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

LHX2 haploinsufficiency causes a variable neurodevelopmental disorder.

PURPOSE: LHX2 encodes the LIM homeobox 2 transcription factor (LHX2), which is highly expressed in brain and well conserved across species, but it has not been clearly linked to neurodevelopmental disorders (NDDs) to date. METHODS: Through international collaboration, we identified 19 individuals from 18 families with variable neurodevelopmental phenotypes, carrying a small chromosomal deletion, likely gene-disrupting or missense variants in LHX2. Functional consequences of missense variants were investigated in cellular systems. RESULTS: Affected individuals presented with developmental and/or behavioral abnormalities, autism spectrum disorder, variable intellectual disability, and microcephaly. We observed nucleolar accumulation for 2 missense variants located within the DNA-binding HOX domain, impaired interaction with co-factor LDB1 for another variant located in the protein-protein interaction-mediating LIM domain, and impaired transcriptional activation by luciferase assay for 4 missense variants. CONCLUSION: We implicate LHX2 haploinsufficiency by deletion and likely gene-disrupting variants as causative for a variable NDD. Our findings suggest a loss-of-function mechanism also for likely pathogenic LHX2 missense variants. Together, our observations underscore the importance of LHX2 in the nervous system and for variable neurodevelopmental phenotypes.

Recurrent FOXP4 nonsense variant in two unrelated patients: Association with neurodevelopmental disease and congenital diaphragmatic hernia.

De novo variants in FOXP4 were recently associated with a neurodevelopmental disorder characterized by speech and language delay, growth abnormalities, hypotonia, and variable congenital abnormalities, including congenital diaphragmatic hernia, cervical spine abnormalities, strabismus, cryptorchidism, and ptosis. The variant spectrum in this small cohort was limited to de novo missense except for one frameshift, the inheritance of which was unknown. Variants tested in vitro exhibited reduced repressor transcriptional activity, indicating loss of function is the likely mechanism of disease, but only one frameshift variant was reported. Here, we report four affected individuals from two unrelated families heterozygous for a nonsense variant, c.1893C > G, p.Tyr631*, in FOXP4. The phenotype of the affected children includes developmental delay, feeding difficulties in infancy, and similar facial features. In both cases, the variant was inherited from a parent with mild or even subclinical features. Interestingly, one patient presented with congenital diaphragmatic hernia, as reported in two other FOXP4 patients. This report implicates FOXP4 truncating variants in human disease and highlights the wide phenotypic spectrum and variable expressivity.

Novel blended SNRPE-related spliceosomopathy phenotype characterized by microcephaly and congenital atrichia.

Variants in genes encoding core components of the spliceosomes are associated with craniofacial syndromes, collectively called craniofacial spliceosomopathies. SNRPE encodes a core component of pre-mRNA processing U-rich small nuclear ribonuclear proteins (UsnRNPs). Heterozygous variants in SNRPE have been reported in six families with isolated hypotrichosis simplex in addition to one case of isolated non syndromic congenital microcephaly. Here, we report a patient with a novel blended phenotype of microcephaly and congenital atrichia with multiple congenital anomalies due to a de novo intronic SNRPE deletion, c.82-28_82-16del, which results in exon skipping. As discussed within, this phenotype, which we propose be named SNRPE-related syndromic microcephaly and hypotrichosis, overlaps other craniofacial splicesosomopathies.

Functionally impaired RPL8 variants associated with Diamond-Blackfan anemia and a Diamond-Blackfan anemia-like phenotype.

Diamond-Blackfan anemia is a rare genetic disease characterized by erythroblastopenia and a large spectrum of developmental anomalies. The vast majority of the cases genetically described are linked to heterozygous pathogenic variants in more than 20 ribosomal protein genes. Here we report an atypical clinical case of DBA associated with a missense variant in RPL8, which encodes RPL8/uL2, a protein of the 60S large ribosomal subunit. RPL8 has been previously implicated as a candidate disease gene in one patient with DBA bearing another type of missense variant; however, evidence for pathogenicity was limited to computational tools. Using functional studies in lymphoblastoid cells as well as yeast models, we show that the RPL8 variants detected in these two patients encode functionally deficient proteins that affect ribosome production and are therefore likely pathogenic. We propose to include RPL8 in the list of DBA-associated genes.

Haploinsufficiency of the Notch Ligand DLL1 Causes Variable Neurodevelopmental Disorders.

Notch signaling is an established developmental pathway for brain morphogenesis. Given that Delta-like 1 (DLL1) is a ligand for the Notch receptor and that a few individuals with developmental delay, intellectual disability, and brain malformations have microdeletions encompassing DLL1, we hypothesized that insufficiency of DLL1 causes a human neurodevelopmental disorder. We performed exome sequencing in individuals with neurodevelopmental disorders. The cohort was identified using known Matchmaker Exchange nodes such as GeneMatcher. This method identified 15 individuals from 12 unrelated families with heterozygous pathogenic DLL1 variants (nonsense, missense, splice site, and one whole gene deletion). The most common features in our cohort were intellectual disability, autism spectrum disorder, seizures, variable brain malformations, muscular hypotonia, and scoliosis. We did not identify an obvious genotype-phenotype correlation. Analysis of one splice site variant showed an in-frame insertion of 12 bp. In conclusion, heterozygous DLL1 pathogenic variants cause a variable neurodevelopmental phenotype and multi-systemic features. The clinical and molecular data support haploinsufficiency as a mechanism for the pathogenesis of this DLL1-related disorder and affirm the importance of DLL1 in human brain development.

Clinical Validation of Genome Reference Consortium Human Build 38 in a Laboratory Utilizing Next-Generation Sequencing Technologies.

BACKGROUND: Laboratories utilizing next-generation sequencing align sequence data to a standardized human reference genome (HRG). Several updated versions, or builds, have been released since the original HRG in 2001, including the Genome Reference Consortium Human Build 38 (GRCh38) in 2013. However, most clinical laboratories still use GRCh37, which was released in 2009. We report our laboratory's clinical validation of GRCh38. METHODS: Migration to GRCh38 was validated by comparing the coordinates (lifting over) of 9443 internally curated variants from GRCh37 to GRCh38, globally comparing protein coding sequence variants aligned with GRCh37 vs GRCh38 from 917 exomes, assessing genes with known discrepancies, comparing coverage differences, and establishing the analytic sensitivity and specificity of variant detection using Genome in a Bottle data. RESULTS: Eight discrepancies, due to strand swap or reference base, were observed. Three clinically relevant variants had the GRCh37 alternate allele as the reference allele in GRCh38. A comparison of 88 295 calls between builds identified 8 disease-associated genes with sequence differences: ABO, BNC2, KIZ, NEFL, NR2E3, PTPRQ, SHANK2, and SRD5A2. Discrepancies in coding regions in GRCh37 were resolved in GRCh38. CONCLUSIONS: There were a small number of clinically significant changes between the 2 genome builds. GRCh38 provided improved detection of nucleotide changes due to the resolution of discrepancies present in GRCh37. Implementation of GRCh38 results in more accurate and consistent reporting.

Phenotypic expansion and variable expressivity in individuals with JARID2-related intellectual disability: A case series.

Loss of function variants in JARID2 were recently reported in 16 patients with a neurodevelopmental disorder characterized by delays, intellectual and learning disability, autism, behavioral abnormalities, and dysmorphic features. Most cases were de novo, with only one variant inherited from an affected parent. Here, we present seven additional individuals from five families with pathogenic or likely pathogenic JARID2 variants, confirming this gene-disease association and highlighting palatal abnormalities and heart defects as part of the phenotype. In addition, we report inheritance of JARID2 variants from mildly affected parents, demonstrating the variable expressivity of the disease. We also note the high prevalence of intragenic JARID2 copy number variants, emphasizing the importance of exon-level analysis.

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

Dual Molecular Effects of Dominant RORA Mutations Cause Two Variants of Syndromic Intellectual Disability with Either Autism or Cerebellar Ataxia.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

Challenges in genetic testing: clinician variant interpretation processes and the impact on clinical care.

PURPOSE: Efforts have been made to standardize laboratory variant interpretation, but clinicians are ultimately tasked with clinical correlation and application of genetic test results in patient care. This study aimed to explore processes clinicians utilize when reviewing and returning genetic test results, and how they impact patient care. METHODS: Medical geneticists, genetic counselors, and nongenetics clinicians from two Midwestern states completed surveys (n = 98) and in-depth interviews (n = 29) on practices of reviewing and returning genetic test results. Retrospective chart review (n = 130) examined discordant interpretations and the impact on care. RESULTS: Participants reported variable behaviors in both reviewing and returning results based on factors such as confidence, view of role, practice setting, and relationship with the lab. Providers did not report requesting changes to variant classifications from laboratories, but indicated relaying conflicting classifications to patients in some cases. Chart reviews revealed medically impactful differences in interpretation between laboratories and clinicians in 18 (13.8%) records. CONCLUSION: Clinician practices for reviewing and integrating genetic test results into patient care vary within and between specialties and impact patient care. Strategies to better incorporate both laboratory and clinician expertise into interpretation of genetic results could result in improved care across providers and settings.

Nager syndrome in patient lacking acrofacial dysostosis: Expanding the phenotypic spectrum of SF3B4-related disease.

Nager syndrome epitomizes the acrofacial dysostoses, which are characterized by craniofacial and limb defects. The craniofacial defects include midfacial retrusion, downslanting palpebral fissures, prominent nasal bridge, and micrognathia. Limb malformations typically include hypoplasia or aplasia of radial elements including the thumb. Nager syndrome is caused by haploinsufficiency of SF3B4, encoding a spliceosomal protein called SAP49. Here, we report a patient with a loss of function variant in SF3B4 without acrofacial dysostosis or limb defects, whose reason for referral was developmental and growth delay. This patient is evidence of a broader phenotypic spectrum associated with SF3B4 variants than previously appreciated.

Delayed diagnosis of holocarboxylase synthetase deficiency in three patients with prominent skin findings.

Holocarboxylase deficiency (HLCSD) is caused by biallelic pathogenic variants in HLCS and is associated with poor feeding, emesis, lethargy, seizures, life-threatening metabolic acidosis, and hyperammonemia. Skin involvement in HLCSD is typically described as scaly, erythrodermic, seborrhea-like, or ichthyosiform, but there is a paucity of reports. We report three patients, including two siblings, with HLCSD and significant cutaneous manifestations including ichthyosiform dermatitis and a presentation with features of annular pustular psoriasis. In this report, we show that persistent, unexplained rash, even in the absence of other clinical findings, should warrant consideration and potential workup for HLCSD.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

OBJECTIVE: Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. METHODS: We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. RESULTS: Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. INTERPRETATION: Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

De novo heterozygous missense and loss-of-function variants in CDC42BPB are associated with a neurodevelopmental phenotype.

CDC42BPB encodes MRCKß (myotonic dystrophy-related Cdc42-binding kinase beta), a serine/threonine protein kinase, and a downstream effector of CDC42, which has recently been associated with Takenouchi-Kosaki syndrome, an autosomal dominant neurodevelopmental disorder. We identified 12 heterozygous predicted deleterious variants in CDC42BPB (9 missense, 2 frameshift, and 1 nonsense) in 14 unrelated individuals (confirmed de novo in 11/14) with neurodevelopmental disorders including developmental delay/intellectual disability, autism, hypotonia, and structural brain abnormalities including cerebellar vermis hypoplasia and agenesis/hypoplasia of the corpus callosum. The frameshift and nonsense variants in CDC42BPB are expected to be gene-disrupting and lead to haploinsufficiency via nonsense-mediated decay. All missense variants are located in highly conserved and functionally important protein domains/regions: 3 are found in the protein kinase domain, 2 are in the citron homology domain, and 4 in a 20-amino acid sequence between 2 coiled-coil regions, 2 of which are recurrent. Future studies will help to delineate the natural history and to elucidate the underlying biological mechanisms of the missense variants leading to the neurodevelopmental and behavioral phenotypes.

Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αß-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective ß-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/ß-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

Clinical genome sequencing in an unbiased pediatric cohort.

PURPOSE: We report for the first time, the use of clinical genome sequencing (GS) in an unbiased pediatric cohort. We describe the clinical validation, patient metrics, ordering patterns, results, reimbursement, and physician retrieval of results for the first consecutive 80 cases. METHODS: Clinical GS was performed for both inpatients and outpatients undergoing etiologic evaluations. Results were reported in the electronic medical record. Evidence of report retrieval by clinicians and whether interpretation was concordant with laboratory report was obtained through retrospective chart review. RESULTS: Twenty definitive diagnoses were made in 19 patients (24%; n = 80). Except for two partial gene deletions, all diagnostic variants would have been detectable by our exome methods. Surprisingly, there was no documentation of communication of results to the family in the medical record for 17.5% of patients, and in 7.5%, physician and laboratory interpretations were discordant. Average insurance reimbursement was 30.2%, with yield for commercial payers significantly higher, at 54.1%. CONCLUSIONS: The detection rate of GS is equivalent and potentially superior to exome sequencing (ES). Reimbursement rates were variable but overall satisfactory for commercial insurers, and poor for government entities. In addition, we identify opportunities for improvement in the communication of results to families, likely translatable to other tests and other institutions.