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.

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.

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.

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.

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.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Assessing performance of pathogenicity predictors using clinically relevant variant datasets.

BACKGROUND: Pathogenicity predictors are integral to genomic variant interpretation but, despite their widespread usage, an independent validation of performance using a clinically relevant dataset has not been undertaken. METHODS: We derive two validation datasets: an 'open' dataset containing variants extracted from publicly available databases, similar to those commonly applied in previous benchmarking exercises, and a 'clinically representative' dataset containing variants identified through research/diagnostic exome and panel sequencing. Using these datasets, we evaluate the performance of three recent meta-predictors, REVEL, GAVIN and ClinPred, and compare their performance against two commonly used in silico tools, SIFT and PolyPhen-2. RESULTS: Although the newer meta-predictors outperform the older tools, the performance of all pathogenicity predictors is substantially lower in the clinically representative dataset. Using our clinically relevant dataset, REVEL performed best with an area under the receiver operating characteristic curve of 0.82. Using a concordance-based approach based on a consensus of multiple tools reduces the performance due to both discordance between tools and false concordance where tools make common misclassification. Analysis of tool feature usage may give an insight into the tool performance and misclassification. CONCLUSION: Our results support the adoption of meta-predictors over traditional in silico tools, but do not support a consensus-based approach as in current practice.

Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

Novel nonsense variants in SLURP1 and DSG1 cause palmoplantar keratoderma in Pakistani families.

BACKGROUND: Inherited palmoplantar keratodermas (PPKs) are clinically and genetically heterogeneous and phenotypically diverse group of genodermatoses characterized by hyperkeratosis of the palms and soles. More than 20 genes have been reported to be associated with PPKs including desmoglein 1 (DSG1) a key molecular component for epidermal adhesion and differentiation. Mal de Meleda (MDM) is a rare inherited autosomal recessive genodermatosis characterized by transgrediens PPK, associated with mutations in the secreted LY6/PLAUR domain containing 1 (SLURP1) gene. METHODS: This study describes clinical as well as genetic whole exome sequencing (WES) and di-deoxy sequencing investigations in two Pakistani families with a total of 12 individuals affected by PPK. RESULTS: WES identified a novel homozygous nonsense variant in SLURP1, and a novel heterozygous nonsense variant in DSG1, as likely causes of the conditions in each family. CONCLUSIONS: This study expands knowledge regarding the molecular basis of PPK, providing important information to aid clinical management in families with PPK from Pakistan.

CLCC1 c. 75C>A Mutation in Pakistani Derived Retinitis Pigmentosa Families Likely Originated With a Single Founder Mutation 2,000-5,000 Years Ago.

Background: A CLCC1 c. 75C > A (p.D25E) mutation has been associated with autosomal recessive pigmentosa in patients in and from Pakistan. CLCC1 is ubiquitously expressed, and knockout models of this gene in zebrafish and mice are lethal in the embryonic period, suggesting that possible retinitis pigmentosa mutations in this gene might be limited to those leaving partial activity. In agreement with this hypothesis, the mutation is the only CLCC1 mutation associated with retinitis pigmentosa to date, and all identified patients with this mutation share a common SNP haplotype surrounding the mutation, suggesting a common founder. Methods: SNPs were genotyped by a combination of WGS and Sanger sequencing. The original founder haplotype, and recombination pathways were delineated by examination to minimize recombination events. Mutation age was estimated by four methods including an explicit solution, an iterative approach, a Bayesian approach and an approach based solely on ancestral segment lengths using high density SNP data. Results: All members of each of the nine families studied shared a single autozygous SNP haplotype for the CLCC1 region ranging from approximately 1-3.5 Mb in size. The haplotypes shared by the families could be derived from a single putative ancestral haplotype with at most two recombination events. Based on the haplotype and Gamma analysis, the estimated age of the founding mutation varied from 79 to 196 generations, or approximately 2,000-5,000 years, depending on the markers used in the estimate. The DMLE (Bayesian) estimates ranged from 2,160 generations assuming a population growth rate of 0-309 generations assuming a population growth rate of 2% with broad 95% confidence intervals. Conclusion: These results provide insight into the origin of the CLCC1 mutation in the Pakistan population. This mutation is estimated to have occurred 2000-5,000 years ago and has been transmitted to affected families of Pakistani origin in geographically dispersed locations around the world. This is the only mutation in CLCC1 identified to date, suggesting that the CLCC1 gene is under a high degree of constraint, probably imposed by functional requirements for this gene during embryonic development.

Final Exon Frameshift Biallelic PTPN23 Variants Are Associated with Microcephalic Complex Hereditary Spastic Paraplegia.

The hereditary spastic paraplegias (HSPs) are a large clinically heterogeneous group of genetic disorders classified as 'pure' when the cardinal feature of progressive lower limb spasticity and weakness occurs in isolation and 'complex' when associated with other clinical signs. Here, we identify a homozygous frameshift alteration occurring in the last coding exon of the protein tyrosine phosphatase type 23 (PTPN23) gene in an extended Palestinian family associated with autosomal recessive complex HSP. PTPN23 encodes a catalytically inert non-receptor protein tyrosine phosphatase that has been proposed to interact with the endosomal sorting complex required for transport (ESCRT) complex, involved in the sorting of ubiquitinated cargos for fusion with lysosomes. In view of our data, we reviewed previously published candidate pathogenic PTPN23 variants to clarify clinical outcomes associated with pathogenic gene variants. This determined that a number of previously proposed candidate PTPN23 alterations are likely benign and revealed that pathogenic biallelic PTPN23 alterations cause a varied clinical spectrum comprising of complex HSP associated with microcephaly, which may occur without intellectual impairment or involve more severe neurological disease. Together, these findings highlight the importance of the inclusion of the PTPN23 gene on HSP gene testing panels globally.

Consolidating biallelic SDHD variants as a cause of mitochondrial complex II deficiency.

Isolated mitochondrial complex II deficiency is a rare cause of mitochondrial respiratory chain disease. To date biallelic variants in three genes encoding mitochondrial complex II molecular components have been unequivocally associated with mitochondrial disease (SDHA/SDHB/SDHAF1). Additionally, variants in one further complex II component (SDHD) have been identified as a candidate cause of isolated mitochondrial complex II deficiency in just two unrelated affected individuals with clinical features consistent with mitochondrial disease, including progressive encephalomyopathy and lethal infantile cardiomyopathy. We present clinical and genomic investigations in four individuals from an extended Palestinian family with clinical features consistent with an autosomal recessive mitochondrial complex II deficiency, in which our genomic studies identified a homozygous NM_003002.3:c.[205 G > A];[205 G > A];p.[(Glu69Lys)];[(Glu69Lys)] SDHD variant as the likely cause. Reviewing previously published cases, these findings consolidate disruption of SDHD function as a cause of mitochondrial complex II deficiency and further define the phenotypic spectrum associated with SDHD gene variants.

Dominant mitochondrial membrane protein-associated neurodegeneration (MPAN) variants cluster within a specific C19orf12 isoform.

Mitochondria membrane protein-associated neurodegeneration (MPAN) neurodegenerative disorder is typically associated with biallelic C19orf12 variants. Here we describe a new and review candidate previous monoallelic de novo C19orf12 variants to define loss of function mutations located in the putative non-membrane spanning C19orf12 isoform as the potential basis of monoallelic MPAN.

MNS1 variant associated with situs inversus and male infertility.

Ciliopathy disorders due to abnormalities of motile cilia encompass a range of autosomal recessive conditions typified by chronic otosinopulmonary disease, infertility, situs abnormalities and hydrocephalus. Using a combination of genome-wide SNP mapping and whole exome sequencing (WES), we investigated the genetic cause of a form of situs inversus (SI) and male infertility present in multiple individuals in an extended Amish family, assuming that an autosomal recessive founder variant was responsible. This identified a single shared (2.34 Mb) region of autozygosity on chromosome 15q21.3 as the likely disease locus, in which we identified a single candidate biallelic frameshift variant in MNS1 [NM_018365.2: c.407_410del; p.(Glu136Glyfs*16)]. Genotyping of multiple family members identified randomisation of the laterality defects in other homozygous individuals, with all wild type or MNS1 c.407_410del heterozygous carriers being unaffected, consistent with an autosomal recessive mode of inheritance. This study identifies an MNS1 variant as a cause of laterality defects and male infertility in humans, mirroring findings in Mns1-deficient mice which also display male infertility and randomisation of left-right asymmetry of internal organs, confirming a crucial role for MNS1 in nodal cilia and sperm flagella formation and function.

An Amish founder variant consolidates disruption of CEP55 as a cause of hydranencephaly and renal dysplasia.

The centrosomal protein 55 kDa (CEP55 (OMIM 610000)) plays a fundamental role in cell cycle regulation and cytokinesis. However, the precise role of CEP55 in human embryonic growth and development is yet to be fully defined. Here we identified a novel homozygous founder frameshift variant in CEP55, present at low frequency in the Amish community, in two siblings presenting with a lethal foetal disorder. The features of the condition are reminiscent of a Meckel-like syndrome comprising of Potter sequence, hydranencephaly, and cystic dysplastic kidneys. These findings, considered alongside two recent studies of single families reporting loss of function candidate variants in CEP55, confirm disruption of CEP55 function as a cause of this clinical spectrum and enable us to delineate the cardinal clinical features of this disorder, providing important new insights into early human development.