Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome.

GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Bi-allelic CAMSAP1 variants cause a clinically recognizable neuronal migration disorder.

Non-centrosomal microtubules are essential cytoskeletal filaments that are important for neurite formation, axonal transport, and neuronal migration. They require stabilization by microtubule minus-end-targeting proteins including the CAMSAP family of molecules. Using exome sequencing on samples from five unrelated families, we show that bi-allelic CAMSAP1 loss-of-function variants cause a clinically recognizable, syndromic neuronal migration disorder. The cardinal clinical features of the syndrome include a characteristic craniofacial appearance, primary microcephaly, severe neurodevelopmental delay, cortical visual impairment, and seizures. The neuroradiological phenotype comprises a highly recognizable combination of classic lissencephaly with a posterior more severe than anterior gradient similar to PAFAH1B1(LIS1)-related lissencephaly and severe hypoplasia or absence of the corpus callosum; dysplasia of the basal ganglia, hippocampus, and midbrain; and cerebellar hypodysplasia, similar to the tubulinopathies, a group of monogenic tubulin-associated disorders of cortical dysgenesis. Neural cell rosette lineages derived from affected individuals displayed findings consistent with these phenotypes, including abnormal morphology, decreased cell proliferation, and neuronal differentiation. Camsap1-null mice displayed increased perinatal mortality, and RNAScope studies identified high expression levels in the brain throughout neurogenesis and in facial structures, consistent with the mouse and human neurodevelopmental and craniofacial phenotypes. Together our findings confirm a fundamental role of CAMSAP1 in neuronal migration and brain development and define bi-allelic variants as a cause of a clinically distinct neurodevelopmental disorder in humans and mice.

Biallelic variants in TRAPPC10 cause a microcephalic TRAPPopathy disorder in humans and mice.

The highly evolutionarily conserved transport protein particle (TRAPP) complexes (TRAPP II and III) perform fundamental roles in subcellular trafficking pathways. Here we identified biallelic variants in TRAPPC10, a component of the TRAPP II complex, in individuals with a severe microcephalic neurodevelopmental disorder. Molecular studies revealed a weakened interaction between mutant TRAPPC10 and its putative adaptor protein TRAPPC2L. Studies of patient lymphoblastoid cells revealed an absence of TRAPPC10 alongside a concomitant absence of TRAPPC9, another key TRAPP II complex component associated with a clinically overlapping neurodevelopmental disorder. The TRAPPC9/10 reduction phenotype was recapitulated in TRAPPC10-/- knockout cells, which also displayed a membrane trafficking defect. Notably, both the reduction in TRAPPC9 levels and the trafficking defect in these cells could be rescued by wild type but not mutant TRAPPC10 gene constructs. Moreover, studies of Trappc10-/- knockout mice revealed neuroanatomical brain defects and microcephaly, paralleling findings seen in the human condition as well as in a Trappc9-/- mouse model. Together these studies confirm autosomal recessive TRAPPC10 variants as a cause of human disease and define TRAPP-mediated pathomolecular outcomes of importance to TRAPPC9 and TRAPPC10 mediated neurodevelopmental disorders in humans and mice.

100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

TECPR2-related hereditary sensory and autonomic neuropathy in two siblings from Palestine.

Due to the majority of currently available genome data deriving from individuals of European ancestry, the clinical interpretation of genomic variants in individuals from diverse ethnic backgrounds remains a major diagnostic challenge. Here, we investigated the genetic cause of a complex neurodevelopmental phenotype in two Palestinian siblings. Whole exome sequencing identified a homozygous missense TECPR2 variant (Chr14(GRCh38):g.102425085G>A; NM_014844.5:c.745G>A, p.(Gly249Arg)) absent in gnomAD, segregating appropriately with the inheritance pattern in the family. Variant assessment with in silico pathogenicity prediction and protein modeling tools alongside population database frequencies led to classification as a variant of uncertain significance. As pathogenic TECPR2 variants are associated with hereditary sensory and autonomic neuropathy with intellectual disability, we reviewed previously published candidate TECPR2 missense variants to clarify clinical outcomes and variant classification using current approved guidelines, classifying a number of published variants as of uncertain significance. This work highlights genomic healthcare inequalities and the challenges in interpreting rare genetic variants in populations underrepresented in genomic databases. It also improves understanding of the clinical and genetic spectrum of TECPR2-related neuropathy and contributes to addressing genomic data disparity and inequalities of the genomic architecture in Palestinian populations.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.

'Something that helped the whole picture': Experiences of parents offered rapid prenatal exome sequencing in routine clinical care in the English National Health Service.

OBJECTIVES: In October 2020, rapid prenatal exome sequencing (pES) was introduced into routine National Health Service (NHS) care in England. This study aimed to explore parent experiences and their information and support needs from the perspective of parents offered pES and of health professionals involved in its delivery. METHODS: In this qualitative study, semi-structured interviews were conducted with 42 women and 6 male partners and 63 fetal medicine and genetic health professionals. Interviews were transcribed verbatim and analysed using thematic analysis. RESULTS: Overall views about pES were positive and parents were grateful to be offered the test. Highlighted benefits of pES included the value of the additional information for pregnancy management and planning for future pregnancies. An anxious wait for results was common, often associated with the need to make decisions near to 24 weeks in pregnancy when there are legal restrictions for late termination. Descriptions of dealing with uncertainty were also common, even when results had been returned. Many parents described pES results as informing decision-making around whether or not to terminate pregnancy. Some professionals were concerned that a non-informative result could be overly reassuring and highlighted that careful counselling was needed to ensure parents have a good understanding of what the result means for their pregnancy. Emotional support from professionals was valued; however, some parents felt that post-test support was lacking. CONCLUSION: Parents and professionals welcomed the introduction of pES. Results inform parents' decision-making around the termination of pregnancy. When there are no diagnostic findings or uncertain findings from pES, personalised counselling that considers scans and other tests are crucial. Directing parents to reliable online sources of information and providing emotional support throughout could improve their experiences of care.

A biallelic SNIP1 Amish founder variant causes a recognizable neurodevelopmental disorder.

SNIP1 (Smad nuclear interacting protein 1) is a widely expressed transcriptional suppressor of the TGF-ß signal-transduction pathway which plays a key role in human spliceosome function. Here, we describe extensive genetic studies and clinical findings of a complex inherited neurodevelopmental disorder in 35 individuals associated with a SNIP1 NM_024700.4:c.1097A>G, p.(Glu366Gly) variant, present at high frequency in the Amish community. The cardinal clinical features of the condition include hypotonia, global developmental delay, intellectual disability, seizures, and a characteristic craniofacial appearance. Our gene transcript studies in affected individuals define altered gene expression profiles of a number of molecules with well-defined neurodevelopmental and neuropathological roles, potentially explaining clinical outcomes. Together these data confirm this SNIP1 gene variant as a cause of an autosomal recessive complex neurodevelopmental disorder and provide important insight into the molecular roles of SNIP1, which likely explain the cardinal clinical outcomes in affected individuals, defining potential therapeutic avenues for future research.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

TMEM63C mutations cause mitochondrial morphology defects and underlie hereditary spastic paraplegia.

The hereditary spastic paraplegias (HSP) are among the most genetically diverse of all Mendelian disorders. They comprise a large group of neurodegenerative diseases that may be divided into 'pure HSP' in forms of the disease primarily entailing progressive lower-limb weakness and spasticity, and 'complex HSP' when these features are accompanied by other neurological (or non-neurological) clinical signs. Here, we identified biallelic variants in the transmembrane protein 63C (TMEM63C) gene, encoding a predicted osmosensitive calcium-permeable cation channel, in individuals with hereditary spastic paraplegias associated with mild intellectual disability in some, but not all cases. Biochemical and microscopy analyses revealed that TMEM63C is an endoplasmic reticulum-localized protein, which is particularly enriched at mitochondria-endoplasmic reticulum contact sites. Functional in cellula studies indicate a role for TMEM63C in regulating both endoplasmic reticulum and mitochondrial morphologies. Together, these findings identify autosomal recessive TMEM63C variants as a cause of pure and complex HSP and add to the growing evidence of a fundamental pathomolecular role of perturbed mitochondrial-endoplasmic reticulum dynamics in motor neurone degenerative diseases.

HERC2 deficiency activates C-RAF/MKK3/p38 signalling pathway altering the cellular response to oxidative stress.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases.

Uncovering the burden of hidden ciliopathies in the 100 000 Genomes Project: a reverse phenotyping approach.

BACKGROUND: The 100 000 Genomes Project (100K) recruited National Health Service patients with eligible rare diseases and cancer between 2016 and 2018. PanelApp virtual gene panels were applied to whole genome sequencing data according to Human Phenotyping Ontology (HPO) terms entered by recruiting clinicians to guide focused analysis. METHODS: We developed a reverse phenotyping strategy to identify 100K participants with pathogenic variants in nine prioritised disease genes (BBS1, BBS10, ALMS1, OFD1, DYNC2H1, WDR34, NPHP1, TMEM67, CEP290), representative of the full phenotypic spectrum of multisystemic primary ciliopathies. We mapped genotype data 'backwards' onto available clinical data to assess potential matches against phenotypes. Participants with novel molecular diagnoses and key clinical features compatible with the identified disease gene were reported to recruiting clinicians. RESULTS: We identified 62 reportable molecular diagnoses with variants in these nine ciliopathy genes. Forty-four have been reported by 100K, 5 were previously unreported and 13 are new diagnoses. We identified 11 participants with unreportable, novel molecular diagnoses, who lacked key clinical features to justify reporting to recruiting clinicians. Two participants had likely pathogenic structural variants and one a deep intronic predicted splice variant. These variants would not be prioritised for review by standard 100K diagnostic pipelines. CONCLUSION: Reverse phenotyping improves the rate of successful molecular diagnosis for unsolved 100K participants with primary ciliopathies. Previous analyses likely missed these diagnoses because incomplete HPO term entry led to incorrect gene panel choice, meaning that pathogenic variants were not prioritised. Better phenotyping data are therefore essential for accurate variant interpretation and improved patient benefit.

Unravelling the genetic basis of retinal dystrophies in Pakistani consanguineous families.

BACKGROUND: Retinitis Pigmentosa (RP) is a clinically and genetically progressive retinal dystrophy associated with severe visual impairments and sometimes blindness, the most common syndromic form of which is Usher syndrome (USH). This study aimed to further increase understanding of the spectrum of RP in the Khyber Pakhtunkhwa region of Pakistan. METHODOLOGY: Four consanguineous families of Pashtun ethnic group were investigated which were referred by the local collaborating ophthalmologists. In total 42 individuals in four families were recruited and investigated using whole exome and dideoxy sequencing. Among them, 20 were affected individuals including 6 in both family 1 and 2, 5 in family 3 and 3 in family 4. RESULT: Pathogenic gene variants were identified in all four families, including two in cone dystrophy and RP genes in the same family (PDE6C; c.480delG, p.Asn161ThrfsTer33 and TULP1; c.238 C > T, p.Gln80Ter) with double-homozygous individuals presenting with more severe disease. Other pathogenic variants were identified in MERTK (c.2194C > T, p.Arg732Ter), RHO (c.448G > A, p.Glu150Lys) associated with non-syndromic RP, and MYO7A (c.487G > A, p.Gly163Arg) associated with USH. In addition, the reported variants were of clinical significance as the PDE6C variant was detected novel, whereas TULP1, MERTK, and MYO7A variants were detected rare and first time found segregating with retinal dystrophies in Pakistani consanguineous families. CONCLUSIONS: This study increases knowledge of the genetic basis of retinal dystrophies in families from Pakistan providing information important for genetic testing and diagnostic provision particularly from the Khyber Pakhtunkhwa region.

No association between SCN9A and monogenic human epilepsy disorders.

Many studies have demonstrated the clinical utility and importance of epilepsy gene panel testing to confirm the specific aetiology of disease, enable appropriate therapeutic interventions, and inform accurate family counselling. Previously, SCN9A gene variants, in particular a c.1921A>T p.(Asn641Tyr) substitution, have been identified as a likely autosomal dominant cause of febrile seizures/febrile seizures plus and other monogenic seizure phenotypes indistinguishable from those associated with SCN1A, leading to inclusion of SCN9A on epilepsy gene testing panels. Here we present serendipitous findings of genetic studies that identify the SCN9A c.1921A>T p.(Asn641Tyr) variant at high frequency in the Amish community in the absence of such seizure phenotypes. Together with findings in UK Biobank these data refute an association of SCN9A with epilepsy, which has important clinical diagnostic implications.

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Reclassification of clinically-detected sequence variants: Framework for genetic clinicians and clinical scientists by CanVIG-UK (Cancer Variant Interpretation Group UK).

PURPOSE: Variant classifications may change over time, driven by emergence of fresh or contradictory evidence or evolution in weighing or combination of evidence items. For variant classifications above the actionability threshold, which is classification of likely pathogenic or pathogenic, clinical actions may be irreversible, such as risk-reducing surgery or prenatal interventions. Variant reclassification up or down across the actionability threshold can therefore have significant clinical consequences. Laboratory approaches to variant reinterpretation and reclassification vary widely. METHODS: Cancer Variant Interpretation Group UK is a multidisciplinary network of clinical scientists and genetic clinicians from across the 24 Molecular Diagnostic Laboratories and Clinical Genetics Services of the United Kingdom (NHS) and Republic of Ireland. We undertook surveys, polls, and national meetings of Cancer Variant Interpretation Group UK to evaluate opinions about clinical and laboratory management regarding variant reclassification. RESULTS: We generated a consensus framework on variant reclassification applicable to cancer susceptibility genes and other clinical areas, which provides explicit recommendations for clinical and laboratory management of variant reclassification scenarios on the basis of the nature of the new evidence, the magnitude of evidence shift, and the final classification score. CONCLUSION: In this framework, clinical and laboratory resources are targeted for maximal clinical effect and minimal patient harm, as appropriate to all resource-constrained health care settings.

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities.

PURPOSE: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. METHODS: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. RESULTS: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. CONCLUSION: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

Elucidating the clinical spectrum and molecular basis of HYAL2 deficiency.

PURPOSE: We previously defined biallelic HYAL2 variants causing a novel disorder in 2 families, involving orofacial clefting, facial dysmorphism, congenital heart disease, and ocular abnormalities, with Hyal2 knockout mice displaying similar phenotypes. In this study, we better define the phenotype and pathologic disease mechanism. METHODS: Clinical and genomic investigations were undertaken alongside molecular studies, including immunoblotting and immunofluorescence analyses of variant/wild-type human HYAL2 expressed in mouse fibroblasts, and in silico modeling of putative pathogenic variants. RESULTS: Ten newly identified individuals with this condition were investigated, and they were associated with 9 novel pathogenic variants. Clinical studies defined genotype-phenotype correlations and confirmed a recognizable craniofacial phenotype in addition to myopia, cleft lip/palate, and congenital cardiac anomalies as the most consistent manifestations of the condition. In silico modeling of missense variants identified likely deleterious effects on protein folding. Consistent with this, functional studies indicated that these variants cause protein instability and a concomitant cell surface absence of HYAL2 protein. CONCLUSION: These studies confirm an association between HYAL2 alterations and syndromic cleft lip/palate, provide experimental evidence for the pathogenicity of missense alleles, enable further insights into the pathomolecular basis of the disease, and delineate the core and variable clinical outcomes of the condition.

A recurrent rare intronic variant in CAPN3 alters mRNA splicing and causes autosomal recessive limb-girdle muscular dystrophy-1 in three Pakistani pedigrees.

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1) is an autosomal recessive disorder characterized by progressive weakness of the proximal limb and girdle muscles. Biallelic mutations in CAPN3 are reported frequently to cause LGMDR1. Here, we describe 11 individuals from three unrelated consanguineous families that present with typical features of LGMDR1 that include proximal muscle wasting, weakness of the upper and lower limbs, and elevated serum creatine kinase. Whole-exome sequencing identified a rare homozygous CAPN3 variant near the exon 2 splice donor site that segregates with disease in all three families. mRNA splicing studies showed partial retention of intronic sequence and subsequent introduction of a premature stop codon (NM_000070.3: c.379 + 3A>G; p.Asp128Glyfs*15). Furthermore, we observe reduced CAPN3 expression in primary dermal fibroblasts derived from an affected individual, suggesting instability and/or nonsense-mediated decay of mutation-bearing mRNA. Genome-wide homozygosity mapping and single-nucleotide polymorphism analysis identified a shared haplotype and supports a possible founder effect for the CAPN3 variant. Together, our data extend the mutational spectrum of LGMDR1 and have implications for improved diagnostics for individuals of Pakistani origin.