CAG repeat mosaicism is gene specific in spinocerebellar ataxias.

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXNs and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXNs and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns.

Mono and biallelic variants in HCN2 cause severe neurodevelopmental disorders.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

Lessons from two series by physicians and caregivers' self-reported data in DDX3X-related disorders.

INTRODUCTION AND METHODS: We report two series of individuals with DDX3X variations, one (48 individuals) from physicians and one (44 individuals) from caregivers. RESULTS: These two series include several symptoms in common, with fairly similar distribution, which suggests that caregivers' data are close to physicians' data. For example, both series identified early childhood symptoms that were not previously described: feeding difficulties, mean walking age, and age at first words. DISCUSSION: Each of the two datasets provides complementary knowledge. We confirmed that symptoms are similar to those in the literature and provides more details on feeding difficulties. Caregivers considered that the symptom attention-deficit/hyperactivity disorder were most worrisome. Both series also reported sleep disturbance. Recently, anxiety has been reported in individuals with DDX3X variants. We strongly suggest that attention-deficit/hyperactivity disorder, anxiety, and sleep disorders need to be treated.

Further characterisation of ARX-related disorders in females due to inherited or de novo variants.

The Aristaless-related homeobox (ARX) gene is located on the X chromosome and encodes a transcription factor that is essential for brain development. While the clinical spectrum of ARX-related disorders is well described in males, from X linked lissencephaly with abnormal genitalia syndrome to syndromic and non-syndromic intellectual disability (ID), its phenotypic delineation in females is incomplete. Carrier females in ARX families are usually asymptomatic, but ID has been reported in some of them, as well as in others with de novo variants. In this study, we collected the clinical and molecular data of 10 unpublished female patients with de novo ARX pathogenic variants and reviewed the data of 63 females from the literature with either de novo variants (n=10), inherited variants (n=33) or variants of unknown inheritance (n=20). Altogether, the clinical spectrum of females with heterozygous pathogenic ARX variants is broad: 42.5% are asymptomatic, 16.4% have isolated agenesis of the corpus callosum (ACC) or mild symptoms (learning disabilities, autism spectrum disorder, drug-responsive epilepsy) without ID, whereas 41% present with a severe phenotype (ie, ID or developmental and epileptic encephalopathy (DEE)). The ID/DEE phenotype was significantly more prevalent in females carrying de novo variants (75%, n=15/20) versus in those carrying inherited variants (27.3%, n=9/33). ACC was observed in 66.7% (n=24/36) of females who underwent a brain MRI. By refining the clinical spectrum of females carrying ARX pathogenic variants, we show that ID is a frequent sign in females with this X linked condition.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

Growth charts in DYRK1A syndrome.

DYRK1A Syndrome (OMIM #614104) is caused by pathogenic variations in the DYRK1A gene located on 21q22. Haploinsufficiency of DYRK1A causes a syndrome with global psychomotor delay and intellectual disability. Low birth weight, growth restriction with feeding difficulties, stature insufficiency, and microcephaly are frequently reported. This study aims to create specific growth charts for individuals with DYRK1A Syndrome and identify parameters for size prognosis. Growth parameters were obtained for 92 individuals with DYRK1A Syndrome (49 males vs. 43 females). The data were obtained from pediatric records, parent reporting, and scientific literature. Growth charts for height, weight, body mass index (BMI), and occipitofrontal circumference (OFC) were generated using generalized additive models through R package gamlss. The growth curves include height, weight, and OFC measurements for patients aged 0-5 years. In accordance with the literature, the charts show that individuals are more likely to present intrauterine growth restriction with low birth weight and microcephaly. The growth is then characterized by severe microcephaly, low weight, and short stature. This study proposes growth charts for widespread use in the management of patients with DYRK1A syndrome.

Clinical and genetic keys to cerebellar ataxia due to FGF14 GAA expansions.

BACKGROUND: SCA27B caused by FGF14 intronic heterozygous GAA expansions with at least 250 repeats accounts for 10-60% of cases with unresolved cerebellar ataxia. We aimed to assess the size and frequency of FGF14 expanded alleles in individuals with cerebellar ataxia as compared with controls and to characterize genetic and clinical variability. METHODS: We sized this repeat in 1876 individuals from France sampled for research purposes in this cross-sectional study: 845 index cases with cerebellar ataxia and 324 affected relatives, 475 controls, as well as 119 cases with spastic paraplegia, and 113 with familial essential tremor. FINDINGS: A higher frequency of expanded allele carriers in index cases with ataxia was significant only above 300 GAA repeats (10.1%, n = 85) compared with controls (1.1%, n = 5) (p < 0.0001) whereas GAA250-299 alleles were detected in 1.7% of both groups. Eight of 14 index cases with GAA250-299 repeats had other causal pathogenic variants (4/14) and/or discordance of co-segregation (5/14), arguing against GAA causality. We compared the clinical signs in 127 GAA≥300 carriers to cases with non-expanded GAA ataxia resulting in defining a key phenotype triad: onset after 45 years, downbeat nystagmus, episodic ataxic features including diplopia; and a frequent absence of dysarthria. All maternally transmitted alleles above 100 GAA were unstable with a median expansion of +18 repeats per generation (r2 = 0.44; p < 0.0001). In comparison, paternally transmitted alleles above 100 GAA mostly decreased in size (-15 GAA (r2 = 0.63; p < 0.0001)), resulting in the transmission bias observed in SCA27B pedigrees. INTERPRETATION: SCA27B diagnosis must consider both the phenotype and GAA expansion size. In carriers of GAA250-299 repeats, the absence of documented familial transmission and a presentation deviating from the key SCA27B phenotype, should prompt the search for an alternative cause. Affected fathers have a reduced risk of having affected children, which has potential implications for genetic counseling. FUNDING: This work was supported by the Fondation pour la Recherche Médicale, grant number 13338 to JLM, the Association Connaître les Syndrome Cérébelleux - France (to GS) and by the European Union's Horizon 2020 research and innovation program under grant agreement No 779257 ("SOLVE-RD" to GS). DP holds a Fellowship award from the Canadian Institutes of Health Research (CIHR). SK received a grant (01GM1905C) from the Federal Ministry of Education and Research, Germany, through the TreatHSP network. This work was supported by the Australian Government National Health and Medical Research Council grants (GNT2001513 and MRFF2007677) to MB and PJL.

Delineation of the adult phenotype of Coffin-Siris syndrome in 35 individuals.

Coffin-Siris syndrome (CSS) is a rare multisystemic autosomal dominant disorder. Since 2012, alterations in genes of the SWI/SNF complex were identified as the molecular basis of CSS, studying largely pediatric cohorts. Therefore, there is a lack of information on the phenotype in adulthood, particularly on the clinical outcome in adulthood and associated risks. In an international collaborative effort, data from 35 individuals ≥ 18 years with a molecularly ascertained CSS diagnosis (variants in ARID1B, ARID2, SMARCA4, SMARCB1, SMARCC2, SMARCE1, SOX11, BICRA) using a comprehensive questionnaire was collected. Our results indicate that overweight and obesity are frequent in adults with CSS. Visual impairment, scoliosis, and behavioral anomalies are more prevalent than in published pediatric or mixed cohorts. Cognitive outcomes range from profound intellectual disability (ID) to low normal IQ, with most individuals having moderate ID. The present study describes the first exclusively adult cohort of CSS individuals. We were able to delineate some features of CSS that develop over time and have therefore been underrepresented in previously reported largely pediatric cohorts, and provide recommendations for follow-up.

A Case Report of SYNE1 Deficiency-Mimicking Mitochondrial Disease and the Value of Pangenomic Investigations.

Mitochondrial disorders are characterized by a huge clinical, biochemical, and genetic heterogeneity, which poses significant diagnostic challenges. Several studies report that more than 50% of patients with suspected mitochondrial disease could have a non-mitochondrial disorder. Thus, only the identification of the causative pathogenic variant can confirm the diagnosis. Herein, we describe the diagnostic journey of a family suspected of having a mitochondrial disorder who were referred to our Genetics Department. The proband presented with the association of cerebellar ataxia, COX-negative fibers on muscle histology, and mtDNA deletions. Whole exome sequencing (WES), supplemented by a high-resolution array, comparative genomic hybridization (array-CGH), allowed us to identify two pathogenic variants in the non-mitochondrial SYNE1 gene. The proband and her affected sister were found to be compound heterozygous for a known nonsense variant (c.13258C>T, p.(Arg4420Ter)), and a large intragenic deletion that was predicted to result in a loss of function. To our knowledge, this is the first report of a large intragenic deletion of SYNE1 in patients with cerebellar ataxia (ARCA1). This report highlights the interest in a pangenomic approach to identify the genetic basis in heterogeneous neuromuscular patients with the possible cause of mitochondrial disease. Moreover, even rare copy number variations should be considered in patients with a phenotype suggestive of SYNE1 deficiency.

Huntington's Disease with Small CAG Repeat Expansions.

BACKGROUND: Carriers of small cytosine-adenine-guanine (CAG) repeats below 39 in the HTT gene are traditionally associated with milder Huntington's disease, but their clinical profile has not been extensively studied. OBJECTIVE: To study the phenotype of CAG36-38 repeat carriers. METHODS: We included 35 patients and premanifest carriers of CAG36-38 repeats. We compared clinical and neuropsychological profiles of 11 CAG36-38 patients with 11 matched CAG40-42 patients. In addition, we analyzed 243 CAG36-38 individuals from the ENROLL study to complete the phenotype description. RESULTS: Global cognitive efficiency and performance in different cognitive subdomains were similar in small CAG36-38 and typically CAG40-42 expanded individuals. Chorea as the first symptom was significantly less frequent for CAG36-38 patients (P = 0.04) despite similar total motor scores at first visit. Total motor score at last visit was significantly lower in CAG36-38 carriers (P = 0.003). The similar cognitive and different motor profile of CAG36-38 (n = 243) and CAG40-42 (n = 4675) carriers was confirmed in the ENROLL database. Additionally, clinicians were significantly less confident in diagnosing Huntington's disease (P = 2.4e-8) and diagnosis happened significantly later in CAG36-38 (P = 2.2e-6) despite a similar age at symptom onset (P = 0.29). CONCLUSIONS: We showed that small CAG36-38 expansion carriers had a similar cognitive profile to those with the more common CAG40-42 expansions. These individuals may evade molecular diagnosis because of the absence of chorea rather than because of a low penetrance of symptoms. This finding should encourage neurologists to consider Huntington's disease in cognitively impaired elderly patients without typical chorea and anticipate consequences for genetic counseling in their offspring. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Extreme phenotypic heterogeneity in non-expansion spinocerebellar ataxias.

Although the best-known spinocerebellar ataxias (SCAs) are triplet repeat diseases, many SCAs are not caused by repeat expansions. The rarity of individual non-expansion SCAs, however, has made it difficult to discern genotype-phenotype correlations. We therefore screened individuals who had been found to bear variants in a non-expansion SCA-associated gene through genetic testing, and after we eliminated genetic groups that had fewer than 30 subjects, there were 756 subjects bearing single-nucleotide variants or deletions in one of seven genes: CACNA1A (239 subjects), PRKCG (175), AFG3L2 (101), ITPR1 (91), STUB1 (77), SPTBN2 (39), or KCNC3 (34). We compared age at onset, disease features, and progression by gene and variant. There were no features that reliably distinguished one of these SCAs from another, and several genes-CACNA1A, ITPR1, SPTBN2, and KCNC3-were associated with both adult-onset and infantile-onset forms of disease, which also differed in presentation. Nevertheless, progression was overall very slow, and STUB1-associated disease was the fastest. Several variants in CACNA1A showed particularly wide ranges in age at onset: one variant produced anything from infantile developmental delay to ataxia onset at 64 years of age within the same family. For CACNA1A, ITPR1, and SPTBN2, the type of variant and charge change on the protein greatly affected the phenotype, defying pathogenicity prediction algorithms. Even with next-generation sequencing, accurate diagnosis requires dialogue between the clinician and the geneticist.

Individual perception of environmental factors that influence lower limbs spasticity in inherited spastic paraparesis.

BACKGROUND: Phenotypic variability is a consistent finding in neurogenetics and therefore applicable to hereditary spastic paraparesis. Identifying reasons for this variability is a challenge. We hypothesized that, in addition to genetic modifiers, extrinsic factors influence variability. OBJECTIVES: Our aim was to describe the clinical variability in hereditary spastic paraparesis from the person's perspective. Our goals were to identify individual and environmental factors that influence muscle tone disorders and derive interventions which could improve spasticity. METHODS: This study was based on self-assessments with questions on nominal and ordinal scales completed by participants with hereditary spastic paraparesis. A questionnaire was completed either in-person in the clinic or electronically via lay organization websites. RESULTS: Among the 325 responders, most had SPG4/SPAST (n = 182, 56%) with a mean age at onset of 31.7 (SD 16.7) years and a mean disease duration of 23 (SD 13.6) years at the time of participation. The 2 factors identified as improving spasticity for > 50% of the responders were physiotherapy (193/325, 59%), and superficial warming (172/308, 55%). Half of the responders (n = 164, 50%) performed physical activity at least once a month and up to once a week. Participants who reported physiotherapy as effective were significantly more satisfied with ≥ 3 sessions per week. Psychologically stressful situations (246/319, 77%) and cold temperatures (202/319, 63%) exacerbated spasticity for most participants. CONCLUSION: Participants perceived that physiotherapy reduced spasticity and that the impact of physiotherapy on spasticity was much greater than other medical interventions. Therefore, people should be encouraged to practice physical activity at least 3 times per week. This study reported participants' opinions: in hereditary spastic paraparesis only functional treatments exist, therefore the participant's expertise is of particular importance.

The neurodevelopmental and facial phenotype in individuals with a TRIP12 variant.

Haploinsufficiency of TRIP12 causes a neurodevelopmental disorder characterized by intellectual disability associated with epilepsy, autism spectrum disorder and dysmorphic features, also named Clark-Baraitser syndrome. Only a limited number of cases have been reported to date. We aimed to further delineate the TRIP12-associated phenotype and objectify characteristic facial traits through GestaltMatcher image analysis based on deep-learning algorithms in order to establish a TRIP12 gestalt. 38 individuals between 3 and 66 years (F = 20, M = 18) - 1 previously published and 37 novel individuals - were recruited through an ERN ITHACA call for collaboration. 35 TRIP12 variants were identified, including frameshift (n = 15) and nonsense (n = 6) variants, as well as missense (n = 5) and splice (n = 3) variants, intragenic deletions (n = 4) and two multigene deletions disrupting TRIP12. Though variable in severity, global developmental delay was noted in all individuals, with language deficit most pronounced. About half showed autistic features and susceptibility to obesity seemed inherent to this disorder. A more severe expression was noted in individuals with a missense variant. Facial analysis showed a clear gestalt including deep-set eyes with narrow palpebral fissures and fullness of the upper eyelids, downturned corners of the mouth and large, often low-set ears with prominent earlobes. We report the largest cohort to date of individuals with TRIP12 variants, further delineating the associated phenotype and introducing a facial gestalt. These findings will improve future counseling and patient guidance.

Fast and reliable detection of repeat expansions in spinocerebellar ataxia using exomes.

Usually, molecular diagnosis of spinocerebellar ataxia is based on a step-by-step approach with targeted sizing of four repeat expansions accounting for most dominant cases, then targeted sequencing of other genes. Nowadays, genome sequencing allows detection of most pathogenic variants in a single step. The ExpansionHunter tool can detect expansions in short-read genome sequencing data. Recent studies have shown that ExpansionHunter can also be used to identify repeat expansions in exome sequencing data. We tested ExpansionHunter on spinocerebellar ataxia exomes in a research context as a second-line analysis, after exclusion of main CAG repeat expansions in half of the probands. First, we confirmed the detection of expansions in seven known expansion carriers and then, after targeted analysis of ATXN1, 2, 3 and 7, CACNA1A, TBP, ATN1, NOP56, AR and HTT in 498 exomes, we found 22 additional pathogenic expansions. Comparison with capillary migration sizing in 247 individuals and confirmation of all expanded alleles detected by ExpansionHunter demonstrated that for these loci, sensitivity and specificity reached 100%. ExpansionHunter detected but underestimated the repeat size for larger expansions, and the normal alleles distribution at each locus should be taken into account to detect expansions. Exome combined with ExpansionHunter is reliable to detect repeat expansions in selected loci as first-line analysis in spinocerebellar ataxia.

Intermediate repeat expansions of TBP and STUB1: Genetic modifier or pure digenic inheritance in spinocerebellar ataxias?

PURPOSE: CAG/CAA repeat expansions in TBP>49 are responsible for spinocerebellar ataxia (SCA) type 17 (SCA17). We previously detected cosegregation of STUB1 variants causing SCA48 with intermediate alleles of TBP in 2 families. This cosegregation questions the existence of SCA48 as a monogenic disease. METHODS: We systematically sequenced TBP repeats in 34 probands of dominant ataxia families with STUB1 variants. In addition, we searched for pathogenic STUB1 variants in probands with expanded alleles of TBP>49 (n = 2) or intermediate alleles of TBP≥40 (n = 47). RESULTS: STUB1 variants were found in half of the TBP40-49 cohort. Mirroring this finding, TBP40-49 alleles were detected in 40% of STUB1 probands. The longer the TBP repeat length, the more likely the occurrence of cognitive impairment (P = .0129) and the faster the disease progression until death (P = .0003). Importantly, 13 STUB1 probands presenting with the full SCA48 clinical phenotype had normal TBP37-39 alleles, excluding digenic inheritance as the sole mode. CONCLUSION: We show that intermediate TBP40-49 alleles act as disease modifiers of SCA48 rather than a STUB1/TBP digenic model. This distinction from what has been proposed before has crucial consequences for genetic counseling in SCA48.

IRF2BPL Causes Mild Intellectual Disability Followed by Late-Onset Ataxia.

Background and Objectives: Neurodevelopmental and neurodegenerative disorders have long been considered as different clinical and molecular entities, and only a few genes are known to be involved in both processes. The IRF2BPL (interferon regulatory factor 2 binding protein like) gene was implicated in a severe pediatric phenotype characterized by developmental and epileptic encephalopathy and early regression. In parallel, inherited IRF2BPL variants have been reported in cohorts of patients with late-onset progressive dystonic and ataxic syndrome with few information about the neurodevelopment of these patients. This study aimed to describe both neurodevelopmental and neurodegenerative aspects of the phenotype in adults with IRF2BPL pathogenic variant. Methods: We report here the clinical and molecular data of 18 individuals carrying truncating IRF2BPL variants (identified by either exome or genome sequencing), including a large pedigree of 16 patients presenting with a neurodevelopmental disorder (NDD) associated with late-onset cerebellar ataxia and atrophy. Results: Genome sequencing identified the p.(Gln117*) variant in a large family first assessed for familial ataxia, with multiple individuals presenting with NDD. The p.(Ser313*) variant was identified by exome sequencing in a second family with a young adult patient with NDD without ataxia which was inherited from her asymptomatic mother, suggesting incomplete penetrance of IRF2BPL-linked disorders. Discussion: This study illustrates the importance of neurologic evaluation of adult patients initially diagnosed with NDD to detect a late-onset neurodegenerative condition. Two different disorders may be clinically diagnosed in the same family, when not considering that NDD and late cerebellar changes may be part of the same molecular spectrum such as for IRF2BPL.

OMIXCARE: OMICS technologies solved about 33% of the patients with heterogeneous rare neuro-developmental disorders and negative exome sequencing results and identified 13% additional candidate variants.

Purpose: Patients with rare or ultra-rare genetic diseases, which affect 350 million people worldwide, may experience a diagnostic odyssey. High-throughput sequencing leads to an etiological diagnosis in up to 50% of individuals with heterogeneous neurodevelopmental or malformation disorders. There is a growing interest in additional omics technologies in translational research settings to examine the remaining unsolved cases. Methods: We gathered 30 individuals with malformation syndromes and/or severe neurodevelopmental disorders with negative trio exome sequencing and array comparative genomic hybridization results through a multicenter project. We applied short-read genome sequencing, total RNA sequencing, and DNA methylation analysis, in that order, as complementary translational research tools for a molecular diagnosis. Results: The cohort was mainly composed of pediatric individuals with a median age of 13.7 years (4 years and 6 months to 35 years and 1 month). Genome sequencing alone identified at least one variant with a high level of evidence of pathogenicity in 8/30 individuals (26.7%) and at least a candidate disease-causing variant in 7/30 other individuals (23.3%). RNA-seq data in 23 individuals allowed two additional individuals (8.7%) to be diagnosed, confirming the implication of two pathogenic variants (8.7%), and excluding one candidate variant (4.3%). Finally, DNA methylation analysis confirmed one diagnosis identified by genome sequencing (Kabuki syndrome) and identified an episignature compatible with a BAFopathy in a patient with a clinical diagnosis of Coffin-Siris with negative genome and RNA-seq results in blood. Conclusion: Overall, our integrated genome, transcriptome, and DNA methylation analysis solved 10/30 (33.3%) cases and identified a strong candidate gene in 4/30 (13.3%) of the patients with rare neurodevelopmental disorders and negative exome sequencing results.

De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder.

PURPOSE: KLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20. METHODS: Patients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed. RESULTS: We studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type ß-propeller domain of the KLHL20 protein, which shapes the substrate binding surface. CONCLUSION: Our findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity.

Rare pathogenic variants in WNK3 cause X-linked intellectual disability.

PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had ID with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked ID with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.