Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Collosum, Axon, Cardiac, Ocular, and Genital Defects.

Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Excess of de novo variants in genes involved in chromatin remodelling in patients with marfanoid habitus and intellectual disability.

PURPOSE: Marfanoid habitus (MH) combined with intellectual disability (ID) (MHID) is a clinically and genetically heterogeneous presentation. The combination of array CGH and targeted sequencing of genes responsible for Marfan or Lujan-Fryns syndrome explain no more than 20% of subjects. METHODS: To further decipher the genetic basis of MHID, we performed exome sequencing on a combination of trio-based (33 subjects) or single probands (31 subjects), of which 61 were sporadic. RESULTS: We identified eight genes with de novo variants (DNVs) in at least two unrelated individuals (ARID1B, ATP1A1, DLG4, EHMT1, NFIX, NSD1, NUP205 and ZEB2). Using simulation models, we showed that five genes (DLG4, NFIX, EHMT1, ZEB2 and ATP1A1) met conservative Bonferroni genomewide significance for an excess of the observed de novo point variants. Overall, at least one pathogenic or likely pathogenic variant was identified in 54.7% of subjects (35/64). These variants fell within 27 genes previously associated with Mendelian disorders, including NSD1 and NFIX, which are known to be mutated in overgrowth syndromes. CONCLUSION: We demonstrated that DNVs were enriched in chromatin remodelling (p=2×10-4) and genes regulated by the fragile X mental retardation protein (p=3×10-8), highlighting overlapping genetic mechanisms between MHID and related neurodevelopmental disorders.

De novo mutations in the X-linked TFE3 gene cause intellectual disability with pigmentary mosaicism and storage disorder-like features.

INTRODUCTION: Pigmentary mosaicism (PM) manifests by pigmentation anomalies along Blaschko's lines and represents a clue toward the molecular diagnosis of syndromic intellectual disability (ID). Together with new insights on the role for lysosomal signalling in embryonic stem cell differentiation, mutations in the X-linked transcription factor 3 (TFE3) have recently been reported in five patients. Functional analysis suggested these mutations to result in ectopic nuclear gain of functions. MATERIALS AND METHODS: Subsequent data sharing allowed the clustering of de novo TFE3 variants identified by exome sequencing on DNA extracted from leucocytes in patients referred for syndromic ID with or without PM. RESULTS: We describe the detailed clinical and molecular data of 17 individuals harbouring a de novo TFE3 variant, including the patients that initially allowed reporting TFE3 as a new disease-causing gene. The 12 females and 5 males presented with pigmentation anomalies on Blaschko's lines, severe ID, epilepsy, storage disorder-like features, growth retardation and recognisable facial dysmorphism. The variant was at a mosaic state in at least two male patients. All variants were missense except one splice variant. Eleven of the 13 variants were localised in exon 4, 2 in exon 3, and 3 were recurrent variants. CONCLUSION: This series further delineates the specific storage disorder-like phenotype with PM ascribed to de novo TFE3 mutation in exons 3 and 4. It confirms the identification of a novel X-linked human condition associated with mosaicism and dysregulation within the mechanistic target of rapamycin (mTOR) pathway, as well as a link between lysosomal signalling and human development.

Pathogenic variants in AIMP1 cause pontocerebellar hypoplasia.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is a non-catalytic component of the multi-tRNA synthetase complex which catalyzes the ligation of amino acids to the correct tRNAs. Pathogenic variants in several aminoacyl-tRNA synthetases genes have been linked to various neurological disorders, including leukodystrophies and pontocerebellar hypoplasias (PCH). To date, loss-of-function variants in AIMP1 have been associated with hypomyelinating leukodystrophy-3 (MIM 260600). Here, we report a novel frameshift AIMP1 homozygous variant (c.160delA,p.Lys54Asnfs) in a child with pontocerebellar hypoplasia and simplified gyral pattern, a phenotype not been previously described with AIMP1 variants, thus expanding the phenotypic spectrum. AIMP1 should be included in diagnostic PCH gene panels.

Clinical whole-exome sequencing for the diagnosis of rare disorders with congenital anomalies and/or intellectual disability: substantial interest of prospective annual reanalysis.

PurposeCongenital anomalies and intellectual disability (CA/ID) are a major diagnostic challenge in medical genetics-50% of patients still have no molecular diagnosis after a long and stressful diagnostic "odyssey." Solo clinical whole-exome sequencing (WES) was applied in our genetics center to improve diagnosis in patients with CA/ID.MethodsThis retrospective study examined 416 consecutive tests performed over 3 years to demonstrate the effectiveness of periodically reanalyzing WES data. The raw data from each nonpositive test was reanalyzed at 12 months with the most recent pipeline and in the light of new data in the literature. The results of the reanalysis for patients enrolled in the third year are not yet available.ResultsOf the 416 patients included, data for 156 without a diagnosis were reanalyzed. We obtained 24 (15.4%) additional diagnoses: 12 through the usual diagnostic process (7 new publications, 4 initially misclassified, and 1 copy-number variant), and 12 through translational research by international data sharing. The final yield of positive results was 27.9% through a strict diagnostic approach, and 2.9% through an additional research strategy.ConclusionThis article highlights the effectiveness of periodically combining diagnostic reinterpretation of clinical WES data with translational research involving data sharing for candidate genes.

Expanding the clinical spectrum of recessive truncating mutations of KLHL7 to a Bohring-Opitz-like phenotype.

BACKGROUND: Bohring-Opitz syndrome (BOS) is a rare genetic disorder characterised by a recognisable craniofacial appearance and a typical 'BOS' posture. BOS is caused by sporadic mutations ofASXL1. However, several typical patients with BOS have no molecular diagnosis, suggesting clinical and genetic heterogeneity. OBJECTIVES: To expand the phenotypical spectrum of autosomal recessive variants of KLHL7, reported as causing Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1)-like syndrome. METHODS: We performed whole-exome sequencing in two families with a suspected recessive mode of inheritance. We used the Matchmaker Exchange initiative to identify additional patients. RESULTS: Here, we report six patients with microcephaly, facial dysmorphism, including exophthalmos, nevus flammeus of the glabella and joint contractures with a suspected BOS posture in five out of six patients. We identified autosomal recessive truncating mutations in the KLHL7 gene. KLHL7 encodes a BTB-kelch protein implicated in the cell cycle and in protein degradation by the ubiquitin-proteasome pathway. Recently, biallelic mutations in the KLHL7 gene were reported in four families and associated with CS/CISS1, characterised by clinical features overlapping with our patients. CONCLUSION: We have expanded the clinical spectrum of KLHL7 autosomal recessive variants by describing a syndrome with features overlapping CS/CISS1 and BOS.

Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes.

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.

Helping Bridge the Great Divides: Supporting Nurse Communication at End of Life.

Many, especially newer, nurses lack the experience for nuanced skillful communication at end of life. This study incorporated the End-of-Life Nursing Education Consortium (ELNEC) Core Curriculum Communication module into a nurse residency program and tested for change in attitudes toward end-of-life (EOL) care. Researchers found significant differences in attitudes toward care of the dying before and after the ELNEC training, and between nurses with greater than and less than 5 years experience. Findings support that ELNEC education can significantly impact nurse attitudes about EOL care.

Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of life.

Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclinical seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clinical presentation in the two families, we hypothesized that the observed phenotype was due to mutations in the same gene, and we performed exome sequencing in three affected individuals. Analysis of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease association was confirmed by cosegregation analysis in additional family members. Screening of 68 additional unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one additional subject with compound heterozygous mutations of SLC13A5 and a similar clinical presentation as the index subjects. Mutations affected key residues for sodium binding, which is critical for citrate transport. These findings underline the value of careful clinical characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metabolism in epilepsy.

Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.

PURPOSE: Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS. METHODS: We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested. RESULTS: We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10-5). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10-25), regardless of the phenotype. CONCLUSION: Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions.Genet Med advance online publication 02 February 2017.

Application of whole-exome sequencing to unravel the molecular basis of undiagnosed syndromic congenital neutropenia with intellectual disability.

Neutropenia can be qualified as congenital when of neonatal onset or when associated with extra-hematopoietic manifestations. Overall, 30% of patients with congenital neutropenia (CN) remain without a molecular diagnosis after a multidisciplinary consultation and tedious diagnostic strategy. In the rare situations when neutropenia is identified and associated with intellectual disability (ID), there are few diagnostic hypotheses to test. This retrospective multicenter study reports on a clinically heterogeneous cohort of 10 unrelated patients with CN associated with ID and no molecular diagnosis prior to whole-exome sequencing (WES). WES provided a diagnostic yield of 40% (4/10). The results suggested that in many cases neutropenia and syndromic manifestations could not be assigned to the same molecular alteration. Three sub-groups of patients were highlighted: (i) severe, symptomatic chronic neutropenia, detected early in life, and related to a known mutation in the CN spectrum (ELANE); (ii) mild to moderate benign intermittent neutropenia, detected later, and associated with mutations in genes implicated in neurodevelopmental disorders (CHD2, HUWE1); and (iii) moderate to severe intermittent neutropenia as a probably undiagnosed feature of a newly reported syndrome (KAT6A). Unlike KAT6A, which seems to be associated with a syndromic form of CN, the other reported mutations may not explain the entire clinical picture. Although targeted gene sequencing can be discussed for the primary diagnosis of severe CN, we suggest that performing WES for the diagnosis of disorders associating CN with ID will not only provide the etiological diagnosis but will also pave the way towards personalized care and follow-up. © 2016 Wiley Periodicals, Inc.

PIK3R1 mutations cause syndromic insulin resistance with lipoatrophy.

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.

PI3K/AKT pathway mutations cause a spectrum of brain malformations from megalencephaly to focal cortical dysplasia.

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum.

Compound heterozygous PKHD1 variants cause a wide spectrum of ductal plate malformations.

Ductal plate malformations (DPM) present with a wide phenotypic spectrum comprising Von Meyenburg complexes (VMC), Caroli disease (CD), Caroli syndrome (CS), and autosomal recessive polycystic kidney disease (ARPKD). Variants in PKHD1 are responsible for ARPKD and CS with a high inter- and intra-familial phenotypic variability. Rare familial cases of CD had been reported and exceptional cases of CD are associated with PKHD1 variants. In a family of three siblings presenting with a wide spectrum of severity of DPM, we performed whole exome sequencing and identified two PKHD1 compound heterozygous variants (c.10444G>A; p.Arg3482Cys and c.5521C>T; p.Glu1841Lys), segregating with the symptoms. Two compound heterozygous PKHD1 variants, including one hypomorphic variant, were identified in two other familial cases of DPM with at least one patient presenting with CD. This report widens the phenotypic variability of PKHD1 variants to VMC, and others hepatic bile ducts malformations with inconstant renal phenotype in adults and highlights the important intra-familial phenotypic variability. It also showed that PKHD1 might be a major gene for CD. This work adds an example of the contribution of exome sequencing, not only in the discovery of new genes but also in expanding the phenotypic spectrum of well-known disease-associated genes, using reverse phenotyping.

Congenital neutropenia with retinopathy, a new phenotype without intellectual deficiency or obesity secondary to VPS13B mutations.

Over one hundred VPS13B mutations are reported in Cohen syndrome (CS). Most cases exhibit a homogeneous phenotype that includes intellectual deficiency (ID), microcephaly, facial dysmorphism, slender extremities, truncal obesity, progressive chorioretinal dystrophy, and neutropenia. We report on a patient carrying two VPS13B splicing mutations with an atypical phenotype that included microcephaly, retinopathy, and congenital neutropenia, but neither obesity nor ID. RNA analysis of the IVS34+2T_+3AinsT mutation did not reveal any abnormal splice fragments but mRNA quantification showed a significant decrease in VPS13B expression. RNA sequencing analysis up- and downstream from the IVS57+2T>C mutation showed abnormal splice isoforms. In contrast to patients with typical CS, who express only abnormal VPS13B mRNA and truncated protein, a dose effect of residual normal VPS13B protein possibly explains the incomplete phenotype in the patient. This observation emphasizes that VPS13B analysis should be performed in cases of congenital neutropenia associated with retinopathy, even in the absence of ID, therefore extending the VPS13B phenotype spectrum.

SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function.

Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

mTOR Pathway Somatic Pathogenic Variants in Focal Malformations of Cortical Development: Novel Variants, Topographic Mapping, and Clinical Outcomes.

Background and Objectives: Somatic and germline pathogenic variants in genes of the mammalian target of rapamycin (mTOR) signaling pathway are a common mechanism underlying a subset of focal malformations of cortical development (FMCDs) referred to as mTORopathies, which include focal cortical dysplasia (FCD) type II, subtypes of polymicrogyria, and hemimegalencephaly. Our objective is to screen resected FMCD specimens with mTORopathy features on histology for causal somatic variants in mTOR pathway genes, describe novel pathogenic variants, and examine the variant distribution in relation to neuroimaging, histopathologic classification, and clinical outcomes. Methods: We performed ultra-deep sequencing using a custom HaloPlexHS Target Enrichment kit in DNA from 21 resected fresh-frozen histologically confirmed FCD type II, tuberous sclerosis complex, or hemimegalencephaly specimens. We mapped the variant alternative allele frequency (AAF) across the resected brain using targeted ultra-deep sequencing in multiple formalin-fixed paraffin-embedded tissue blocks. We also functionally validated 2 candidate somatic MTOR variants and performed targeted RNA sequencing to validate a splicing defect associated with a novel DEPDC5 variant. Results: We identified causal mTOR pathway gene variants in 66.7% (14/21) of patients, of which 13 were somatic with AAF ranging between 0.6% and 12.0%. Moreover, the AAF did not predict balloon cell presence. Favorable seizure outcomes were associated with genetically clear resection borders. Individuals in whom a causal somatic variant was undetected had excellent postsurgical outcomes. In addition, we demonstrate pathogenicity of the novel c.4373_4375dupATG and candidate c.7499T>A MTOR variants in vitro. We also identified a novel germline aberrant splice site variant in DEPDC5 (c.2802-1G>C). Discussion: The AAF of somatic pathogenic variants correlated with the topographic distribution, histopathology, and postsurgical outcomes. Moreover, cortical regions with absent histologic FCD features had negligible or undetectable pathogenic variant loads. By contrast, specimens with frank histologic abnormalities had detectable pathogenic variant loads, which raises important questions as to whether there is a tolerable variant threshold and whether surgical margins should be clean, as performed in tumor resections. In addition, we describe 2 novel pathogenic variants, expanding the mTORopathy genetic spectrum. Although most pathogenic somatic variants are located at mutation hotspots, screening the full-coding gene sequence remains necessary in a subset of patients.

MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels.

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon-intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E-07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS.