CAPRIN1 haploinsufficiency causes a neurodevelopmental disorder with language impairment, ADHD and ASD.

We describe an autosomal dominant disorder associated with loss-of-function variants in the Cell cycle associated protein 1 (CAPRIN1; MIM*601178). CAPRIN1 encodes a ubiquitous protein that regulates the transport and translation of neuronal mRNAs critical for synaptic plasticity, as well as mRNAs encoding proteins important for cell proliferation and migration in multiple cell types. We identified 12 cases with loss-of-function CAPRIN1 variants, and a neurodevelopmental phenotype characterized by language impairment/speech delay (100%), intellectual disability (83%), attention deficit hyperactivity disorder (82%) and autism spectrum disorder (67%). Affected individuals also had respiratory problems (50%), limb/skeletal anomalies (50%), developmental delay (42%) feeding difficulties (33%), seizures (33%) and ophthalmologic problems (33%). In patient-derived lymphoblasts and fibroblasts, we showed a monoallelic expression of the wild-type allele, and a reduction of the transcript and protein compatible with a half dose. To further study pathogenic mechanisms, we generated sCAPRIN1+/- human induced pluripotent stem cells via CRISPR-Cas9 mutagenesis and differentiated them into neuronal progenitor cells and cortical neurons. CAPRIN1 loss caused reduced neuronal processes, overall disruption of the neuronal organization and an increased neuronal degeneration. We also observed an alteration of mRNA translation in CAPRIN1+/- neurons, compatible with its suggested function as translational inhibitor. CAPRIN1+/- neurons also showed an impaired calcium signalling and increased oxidative stress, two mechanisms that may directly affect neuronal networks development, maintenance and function. According to what was previously observed in the mouse model, measurements of activity in CAPRIN1+/- neurons via micro-electrode arrays indicated lower spike rates and bursts, with an overall reduced activity. In conclusion, we demonstrate that CAPRIN1 haploinsufficiency causes a novel autosomal dominant neurodevelopmental disorder and identify morphological and functional alterations associated with this disorder in human neuronal models.

Spinocerebellar ataxia 38: structure-function analysis shows ELOVL5 G230V is proteotoxic, conformationally altered and a mutational hotspot.

Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G>T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulphide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.

Identifying phenotypic expansions for congenital diaphragmatic hernia plus (CDH+) using DECIPHER data.

Congenital diaphragmatic hernia (CDH) can occur in isolation or in conjunction with other birth defects (CDH+). A molecular etiology can only be identified in a subset of CDH cases. This is due, in part, to an incomplete understanding of the genes that contribute to diaphragm development. Here, we used clinical and molecular data from 36 individuals with CDH+ who are cataloged in the DECIPHER database to identify genes that may play a role in diaphragm development and to discover new phenotypic expansions. Among this group, we identified individuals who carried putatively deleterious sequence or copy number variants affecting CREBBP, SMARCA4, UBA2, and USP9X. The role of these genes in diaphragm development was supported by their expression in the developing mouse diaphragm, their similarity to known CDH genes using data from a previously published and validated machine learning algorithm, and/or the presence of CDH in other individuals with their associated genetic disorders. Our results demonstrate how data from DECIPHER, and other public databases, can be used to identify new phenotypic expansions and suggest that CREBBP, SMARCA4, UBA2, and USP9X play a role in diaphragm development.

Evidence that FGFRL1 contributes to congenital diaphragmatic hernia development in humans.

Fibroblast growth factor receptor-like 1 (FGFRL1) encodes a transmembrane protein that is related to fibroblast growth factor receptors but lacks an intercellular tyrosine kinase domain. in vitro studies suggest that FGFRL1 inhibits cell proliferation and promotes cell differentiation and cell adhesion. Mice that lack FGFRL1 die shortly after birth from respiratory distress and have abnormally thin diaphragms whose muscular hypoplasia allows the liver to protrude into the thoracic cavity. Haploinsufficiency of FGFRL1 has been hypothesized to contribute to the development of congenital diaphragmatic hernia (CDH) associated with Wolf-Hirschhorn syndrome. However, data from both humans and mice suggest that disruption of one copy of FGFRL1 alone is insufficient to cause diaphragm defects. Here we report a female fetus with CDH whose 4p16.3 deletion allows us to refine the Wolf-Hirschhorn syndrome CDH critical region to an approximately 1.9 Mb region that contains FGFRL1. We also report a male infant with isolated left-sided diaphragm agenesis who carried compound heterozygous missense variants in FGFRL1. These cases provide additional evidence that deleterious FGFRL1 variants may contribute to the development of CDH in humans.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Congenital Sensorineural Hearing Loss and Inborn Pigmentary Disorders: First Report of Multilocus Syndrome in Piebaldism.

Congenital sensorineural hearing loss may occur in association with inborn pigmentary defects of the iris, hair, and skin. These conditions, named auditory-pigmentary disorders (APDs), represent extremely heterogeneous hereditary diseases, including Waardenburg syndromes, oculocutaneous albinism, Tietz syndrome, and piebaldism. APDs are part of the neurocristopathies, a group of congenital multisystem disorders caused by an altered development of the neural crest cells, multipotent progenitors of a wide variety of different lineages, including those differentiating into peripheral nervous system glial cells and melanocytes. We report on clinical and genetic findings of two monozygotic twins from a large Albanian family who showed a complex phenotype featured by sensorineural congenital deafness, severe neuropsychiatric impairment, and inborn pigmentary defects of hair and skin. The genetic analyzes identified, in both probands, an unreported co-occurrence of a new heterozygous germline pathogenic variant (c.2484 + 5G > T splicing mutation) in the KIT gene, consistent with the diagnosis of piebaldism, and a heterozygous deletion at chromosome 15q13.3, responsible for the neuropsychiatric impairment. This case represents the first worldwide report of dual locus inherited syndrome in piebald patients affected by a complex auditory-pigmentary multisystem phenotype. Here we also synthesize the clinical and genetic findings of all known neurocristopathies characterized by a hypopigmentary congenital disorder.

Docosahexaenoic acid is a beneficial replacement treatment for spinocerebellar ataxia 38.

OBJECTIVE: Spinocerebellar ataxia 38 (SCA38) is caused by mutations in the ELOVL5 gene, which encodes an elongase involved in the synthesis of polyunsaturated fatty acids, including docosahexaenoic acid (DHA). As a consequence, DHA is significantly reduced in the serum of SCA38 subjects. In the present study, we evaluated the safety of DHA supplementation, its efficacy for clinical symptoms, and changes of brain functional imaging in SCA38 patients. METHODS: We enrolled 10 SCA38 patients, and carried out a double-blind randomized placebo-controlled study for 16 weeks, followed by an open-label study with overall 40-week DHA treatment. At baseline and at follow-up visit, patients underwent standardized clinical assessment, brain 18-fluorodeoxyglucose positron emission tomography, electroneurography, and ELOVL5 expression analysis. RESULTS: After 16 weeks, we showed a significant pre-post clinical improvement in the DHA group versus placebo, using the Scale for the Assessment and Rating of Ataxia (SARA; mean difference [MD] = +2.70, 95% confidence interval [CI] = +0.13 to + 5.27, p = 0.042). At 40-week treatment, clinical improvement was found significant by both SARA (MD = +2.2, 95% CI = +0.93 to + 3.46, p = 0.008) and International Cooperative Ataxia Rating Scale (MD = +3.8, 95% CI = +1.39 to + 6.41, p = 0.02) scores; clinical data were corroborated by significant improvement of cerebellar hypometabolism (statistical parametric mapping analyses, false discovery rate corrected). We also showed a decreased expression of ELOVL5 in patients' blood at 40 weeks as compared to baseline. No side effect was recorded. INTERPRETATION: DHA supplementation is a safe and effective treatment for SCA38, showing an improvement of clinical symptoms and cerebellar hypometabolism. Ann Neurol 2017;82:615-621.

A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

ELOVL5 mutations cause spinocerebellar ataxia 38.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples.

Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array-Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR-associated cerebellar hypoplasia (VLDLR-CH) is characterized by non-progressive congenital ataxia and moderate-to-profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array-CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing. © 2016 Wiley Periodicals, Inc.

A novel 3q29 deletion associated with autism, intellectual disability, psychiatric disorders, and obesity.

Copy number variation (CNV) has been associated with a variety of neuropsychiatric disorders, including intellectual disability/developmental delay (ID/DD), autism spectrum disorder (ASD), and schizophrenia (SCZ). Often, individuals carrying the same pathogenic CNV display high clinical variability. By array-CGH analysis, we identified a novel familial 3q29 deletion (1.36 Mb), centromeric to the 3q29 deletion region, which manifests with variable expressivity. The deletion was identified in a 3-year-old girl diagnosed with ID/DD and autism and segregated in six family members, all affected by severe psychiatric disorders including schizophrenia, major depression, anxiety disorder, and personality disorder. All individuals carrying the deletion were overweight or obese, and anomalies compatible with optic atrophy were observed in three out of four cases examined. Amongst the 10 genes encompassed by the deletion, the haploinsufficiency of Optic Atrophy 1 (OPA1), associated with autosomal dominant optic atrophy, is likely responsible for the ophthalmological anomalies. We hypothesize that the haploinsufficiency of ATPase type 13A4 (ATP13A4) and/or Hairy/Enhancer of Split Drosophila homolog 1 (HES1) contribute to the neuropsychiatric phenotype, while HES1 deletion might underlie the overweight/obesity. In conclusion, we propose a novel contiguous gene syndrome due to a proximal 3q29 deletion variably associated with autism, ID/DD, psychiatric traits and overweight/obesity.

Array-Comparative Genomic Hybridization Analysis in Fetuses with Major Congenital Malformations Reveals that 24% of Cases Have Pathogenic Deletions/Duplications.

Karyotyping and aCGH are routinely used to identify genetic determinants of major congenital malformations (MCMs) in fetal deaths or terminations of pregnancy after prenatal diagnosis. Pathogenic rearrangements are found with a variable rate of 9-39% for aCGH. We collected 33 fetuses, 9 with a single MCM and 24 with MCMs involving 2-4 organ systems. aCGH revealed copy number variants in 14 out of 33 cases (42%). Eight were classified as pathogenic which account for a detection rate of 24% (8/33) considering fetuses with 1 or more MCMs and 33% (8/24) taking into account fetuses with multiple malformations only. Three of the pathogenic variants were known microdeletion syndromes (22q11.21 deletion, central chromosome 22q11.21 deletion, and TAR syndrome) and 5 were large rearrangements, adding up to >11 Mb per subject and comprising strong phenotype-related genes. One of those was a de novo complex rearrangement, and the remaining 4 duplications and 2 deletions were 130-900 kb in size, containing 1-7 genes, and were classified as variants of unknown clinical significance. Our study confirms aCGH as a powerful technique to ascertain the genetic etiology of fetal major congenital malformations.

An atypical form of AOA2 with myoclonus associated with mutations in SETX and AFG3L2.

BACKGROUND: Hereditary ataxias are a heterogeneous group of neurodegenerative disorders, where exome sequencing may become an important diagnostic tool to solve clinically or genetically complex cases. METHODS: We describe an Italian family in which three sisters were affected by ataxia with postural/intentional myoclonus and involuntary movements at onset, which persisted during the disease. Oculomotor apraxia was absent. Clinical and genetic data did not allow us to exclude autosomal dominant or recessive inheritance and suggest a disease gene. RESULTS: Exome sequencing identified a homozygous c.6292C > T (p.Arg2098*) mutation in SETX and a heterozygous c.346G > A (p.Gly116Arg) mutation in AFG3L2 shared by all three affected individuals. A fourth sister (II.7) had subclinical myoclonic jerks at proximal upper limbs and perioral district, confirmed by electrophysiology, and carried the p.Gly116Arg change. Three siblings were healthy. Pathogenicity prediction and a yeast-functional assay suggested p.Gly116Arg impaired m-AAA (ATPases associated with various cellular activities) complex function. CONCLUSIONS: Exome sequencing is a powerful tool in identifying disease genes. We identified an atypical form of Ataxia with Oculoapraxia type 2 (AOA2) with myoclonus at onset associated with the c.6292C > T (p.Arg2098*) homozygous mutation. Because the same genotype was described in six cases from a Tunisian family with a typical AOA2 without myoclonus, we speculate this latter feature is associated with a second mutated gene, namely AFG3L2 (p.Gly116Arg variant). We suggest that variant phenotypes may be due to the combined effect of different mutated genes associated to ataxia or related disorders, that will become more apparent as the costs of exome sequencing progressively will reduce, amplifying its diagnostics use, and meanwhile proposing significant challenges in the interpretation of the data.

Defining the phenotype associated with microduplication reciprocal to Sotos syndrome microdeletion.

NSD1 point mutations, submicroscopic deletions and intragenic deletions are the major cause of Sotos syndrome, characterized by pre-postnatal generalized overgrowth with advanced bone age, learning disability, seizures, distinctive facial phenotype. Reverse clinical phenotype due to 5q35 microduplication encompassing NSD1 gene has been reported so far in 27 cases presenting with delayed bone age, microcephaly, failure to thrive and seizures in some cases, further supporting a gene dosage effect of NSD1 on growth regulation and neurological functions. Here we depict the clinical presentation of three new cases with 5q35 microduplication outlining a novel syndrome characterized by microcephaly, short stature, developmental delay and in some cases delayed bone maturation, without any typical facial or osseous anomalies.

A de novo X;8 translocation creates a PTK2-THOC2 gene fusion with THOC2 expression knockdown in a patient with psychomotor retardation and congenital cerebellar hypoplasia.

BACKGROUND AND AIM: We identified a balanced de novo translocation involving chromosomes Xq25 and 8q24 in an eight year-old girl with a non-progressive form of congenital ataxia, cognitive impairment and cerebellar hypoplasia. METHODS AND RESULTS: Breakpoint definition showed that the promoter of the Protein Tyrosine Kinase 2 (PTK2, also known as Focal Adhesion Kinase, FAK) gene on chromosome 8q24.3 is translocated 2 kb upstream of the THO complex subunit 2 (THOC2) gene on chromosome Xq25. PTK2 is a well-known non-receptor tyrosine kinase whereas THOC2 encodes a component of the evolutionarily conserved multiprotein THO complex, involved in mRNA export from nucleus. The translocation generated a sterile fusion transcript under the control of the PTK2 promoter, affecting expression of both PTK2 and THOC2 genes. PTK2 is involved in cell adhesion and, in neurons, plays a role in axonal guidance, and neurite growth and attraction. However, PTK2 haploinsufficiency alone is unlikely to be associated with human disease. Therefore, we studied the role of THOC2 in the CNS using three models: 1) THOC2 ortholog knockout in C.elegans which produced functional defects in specific sensory neurons; 2) Thoc2 knockdown in primary rat hippocampal neurons which increased neurite extension; 3) Thoc2 knockdown in neuronal stem cells (LC1) which increased their in vitro growth rate without modifying apoptosis levels. CONCLUSION: We suggest that THOC2 can play specific roles in neuronal cells and, possibly in combination with PTK2 reduction, may affect normal neural network formation, leading to cognitive impairment and cerebellar congenital hypoplasia.

Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome.

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17-74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.

DNA methylation analysis in patients with neurodevelopmental disorders improves variant interpretation and reveals complexity.

Analysis of genomic DNA methylation by generating epigenetic signature profiles (episignatures) is increasingly being implemented in genetic diagnosis. Here we report our experience using episignature analysis to resolve both uncomplicated and complex cases of neurodevelopmental disorders (NDDs). We analyzed 97 NDDs divided into (1) a validation cohort of 59 patients with likely pathogenic/pathogenic variants characterized by a known episignature and (2) a test cohort of 38 patients harboring variants of unknown significance or unidentified variants. The expected episignature was obtained in most cases with likely pathogenic/pathogenic variants (53/59 [90%]), a revealing exception being the overlapping profile of two SMARCB1 pathogenic variants with ARID1A/B:c.6200, confirmed by the overlapping clinical features. In the test cohort, five cases showed the expected episignature, including (1) novel pathogenic variants in ARID1B and BRWD3; (2) a deletion in ATRX causing MRXFH1 X-linked mental retardation; and (3) confirmed the clinical diagnosis of Cornelia de Lange (CdL) syndrome in mutation-negative CdL patients. Episignatures analysis of the in BAF complex components revealed novel functional protein interactions and common episignatures affecting homologous residues in highly conserved paralogous proteins (SMARCA2 M856V and SMARCA4 M866V). Finally, we also found sex-dependent episignatures in X-linked disorders. Implementation of episignature profiling is still in its early days, but with increasing utilization comes increasing awareness of the capacity of this methodology to help resolve the complex challenges of genetic diagnoses.

Analysis of LMNB1 duplications in autosomal dominant leukodystrophy provides insights into duplication mechanisms and allele-specific expression.

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients' fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels.

Bilaterally cleft lip and bilateral thumb polydactyly with triphalangeal component in a patient with two de novo deletions of HSA 4q32 and 4q34 involving PDGFC, GRIA2, and FBXO8 genes.

We report on a newborn boy with a bilateral cleft of the primary palate, duplicated triphalangeal thumbs, and a patent foramen ovale. During childhood he had moderate developmental delay. Brain MRI at 4 years was normal. The concurrence of non-syndromic clefts of the lip/palate (CL/P) and duplicated thumbs with triphalangeal component has, to our knowledge, not been reported so far. In our case, array-CGH analysis documented two de novo deletions (∼1.2 Mb and ∼400 Kb) of the long arm of chromosome 4, containing four genes: platelet-derived growth factor C (PDGFC), glycine receptor beta subunit (GLRB), glutamate receptor ionotropic AMPA2 (GRIA2), and F-box protein 8 gene (FBXO8). PDGFC codes for a mesenchymal cell growth factor already known to be associated with clefts of the lip. Pdgfc(-/-) mice have skeletal anomalies, and facial schisis resembling human cleft/lip palate. GRIA2 codes for a ligand-activated cation channel that mediates the fast component of postsynaptic excitatory currents in neurons, and may be linked to cognitive dysfunction. FBXO8, a gene of unknown function, is a member of the F-box gene family, among which FBXW4, within the minimal duplicated region associated with human split-hand/foot malformation type 3 (SHFM type 3). The presence of overlapping deletions in patients who do not share the same phenotype of our case suggests incomplete penetrance, and a possible effect of modifier genetic factors.