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Neuronal ceroid lipofuscinosis 6 (CLN6) is a rare and fatal autosomal recessive disease primarily affecting the nervous system in children. It is caused by a pathogenic mutation in the CLN6 gene for which no therapy is available. Employing an untargeted metabolomics approach, we analyzed the metabolic changes in CLN6 subjects to see if this system could potentially yield biomarkers for diagnosis and monitoring disease progression. Neuronal-like cells were derived from human fibroblast lines from CLN6-affected subjects (n = 3) and controls (wild type, n = 3). These were used to assess the potential of a neuronal-like cell-based metabolomics approach to identify CLN6 distinctive and specific biomarkers. The most impacted metabolic profile is associated with sphingolipids, glycerophospholipids metabolism, and calcium signaling. Over 2700 spectral features were screened, and fifteen metabolites were identified that differed significantly between both groups, including the sphingolipids C16 GlcCer, C24 GlcCer, C24:1 GlcCer and glycerophospholipids PG 40:6 and PG 40:7. Of note, these fifteen metabolites were downregulated in the CLN6 disease group. This study is the first to analyze the metabolome of neuronal-like cells with a pathogenic mutation in the CLN6 gene and to provide insights into their metabolomic alterations. This could allow for the development of novel biomarkers for monitoring CLN6 disease.

Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4 Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.

We analysed rare variants in the non-coding RNU4-2 gene as a potential cause of neurodevelopmental disorder (NDD) and intellectual disability (ID) in a large cohort of individuals enriched for parental consanguinity.Genome sequencing (GS) data from 22 928 individuals in our Biodatabank were queried for rare, monoallelic variants in RNU4-2. From these, 4918 patients presented with NDD/ID. Human Phenotype Ontology (HPO)-encoded clinical information was extracted and analysed using the ontologyX R package.Nearly 50% of the 4918 patients with NDD/ID reported parental consanguinity. Eight relevant heterozygous RNU4-2 variants were identified in 28 patients. n.64_65insT was the most frequently detected variant (20 patients, 71%), while the remaining variants were found in 1 or 2 patients each (n.65A>G, n.66A>G, n.67A>G, n.70T>C, n.76C>T, n.95C>G and n.135A>C). Four variants are novel or ultra-rare, and two of them are in the 3’ stem loops. HPO-based analysis revealed a consistent syndromic phenotype characterised by NDD, abnormal brain morphology, hypotonia, global developmental delay, microcephaly, seizures, atypical behaviour and facial dysmorphism. RNU4-2 variants accounted for approximately 0.55% of NDD/ID cases in our full cohort, and 0.25% in the subset of consanguineous patients (all genetic causes included).This study underscores the significance of RNU4-2 as a major genetic cause of NDD/ID, extending its relevance to consanguineous patients, where recessive disorders are often suspected. We advocate for the re-evaluation of existing GS data to uncover potential diagnoses and emphasise the importance of GS as a first-tier diagnostic test.

Background Eight different deletions and point variants of the X‐chromosomal gene CNKSR2 have been reported in families with males presenting intellectual disability (ID) and epilepsy. Obligate carrier females with a frameshift variant in the N‐terminal protein coding part of CNKSR2 or with a deletion of the complete gene are not affected. Only for one C‐terminal nonsense variant, two carrier females were mildly affected by seizures without or with mild motor and language delay. Methods Exome sequencing was performed in one female child of a Dutch family, presenting seizures, mild ID, facial dysmorphisms, and abnormalities of the extremities. Potential causative variants were validated by Sanger sequencing. X‐chromosome‐inactivation (XCI) analysis was performed by methylation‐sensitive PCR and fragment‐length analysis of the androgen‐receptor CAG repeat polymorphism. Results We identified a de novo variant, c.2304G>A (p.(Trp768*)), in the C‐terminal protein coding part of the X‐chromosomal gene CNKSR2 in a female patient with seizures and mild ID. Sanger sequencing confirmed the presence of this nonsense variant. XCI analysis showed a mild skewing of X inactivation (20:80) in the blood of our patient. Our variant is the second C‐terminal–affecting CNKSR2 variant described in neurologically affected females. Conclusion Our results indicate that CNKSR2 nonsense variants in the C‐terminal coding part can result in ID with seizures in female variant carriers. We report on a female patient with mild intellectual disability (ID) and seizures and a de novo nonsense variant, p.(Trp768*), in the X‐chromosomal gene CNKSR2. Our variant is the second CNKSR2 variant described in neurologically affected females confirming that CNKSR2 variants can result in ID with seizures in female variant carriers, even though CNKSR2 is subject to X‐inactivation.

To elucidate the novel molecular cause in families with a new autosomal recessive neurodevelopmental disorder. A combination of exome sequencing and gene matching tools was used to identify pathogenic variants in 17 individuals. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and subcellular localization studies were used to characterize gene expression profile and localization. Biallelic variants in the TMEM222 gene were identified in 17 individuals from nine unrelated families, presenting with intellectual disability and variable other features, such as aggressive behavior, shy character, body tremors, decreased muscle mass in the lower extremities, and mild hypotonia. We found relatively high TMEM222 expression levels in the human brain, especially in the parietal and occipital cortex. Additionally, subcellular localization analysis in human neurons derived from induced pluripotent stem cells (iPSCs) revealed that TMEM222 localizes to early endosomes in the synapses of mature iPSC-derived neurons. Our findings support a role for TMEM222 in brain development and function and adds variants in the gene TMEM222 as a novel underlying cause of an autosomal recessive neurodevelopmental disorder.

Intellectual disability (ID), megalencephaly, frontal predominant pachygyria, and seizures, previously called “thin” lissencephaly, are reported to be caused by recessive variants in CRADD. Among five families of different ethnicities identified, one homozygous missense variant, c.509G>A p.(Arg170His), was of Finnish ancestry. Here we report on the phenotypic variability associated for this potential CRADD founder variant in 22 Finnish individuals. Exome sequencing was used to identify candidate genes in Finnish patients presenting with ID. Targeted Sanger sequencing and restriction enzyme analysis were applied to screen for the c.509G>A CRADD variant in cohorts from Finland. Detailed phenotyping and genealogical studies were performed. Twenty two patients were identified with the c.509G>A p.(Arg170His) homozygous variant in CRADD. The majority of the ancestors originated from Northeastern Finland indicating a founder effect. The hallmark of the disease is frontotemporal predominant pachygyria with mild cortical thickening. All patients show ID of variable severity. Aggressive behavior was found in nearly half of the patients, EEG abnormalities in five patients and megalencephaly in three patients. This study provides detailed data about the phenotypic spectrum of patients with lissencephaly due to a CRADD variant that affects function. High inter- and intrafamilial phenotypic heterogeneity was identified in patients with pachygyria caused by the homozygous CRADD founder variant. The phenotype variability suggests that additional genetic and/or environmental factors play a role in the clinical presentation. Since frontotemporal pachygyria is the hallmark of the disease, brain imaging studies are essential to support the molecular diagnosis for individuals with ID and a CRADD variant.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.