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Contains fulltext : 282684.pdf (Publisher’s version ) (Closed access) The pre-mRNA-processing factor 8, encoded by PRPF8, is a scaffolding component of a spliceosome complex involved in the removal of introns from mRNA precursors. Previously, heterozygous pathogenic variants in PRPF8 have been associated with autosomal dominant retinitis pigmentosa. More recently, PRPF8 was suggested as a candidate gene for autism spectrum disorder due to the enrichment of sequence variants in this gene in individuals with neurodevelopmental disorders. We report 14 individuals with various forms of neurodevelopmental conditions, found to have heterozygous, predominantly de novo, missense, and loss-of-function variants in PRPF8. These individuals have clinical features that may represent a new neurodevelopmental syndrome.

CSNK2B has recently been implicated as a disease gene for neurodevelopmental disability (NDD) and epilepsy. Information about developmental outcomes has been limited by the young age and short follow-up for many of the previously reported cases, and further delineation of the spectrum of associated phenotypes is needed. We present 25 new patients with variants in CSNK2B and refine the associated NDD and epilepsy phenotypes. CSNK2B variants were identified by research or clinical exome sequencing, and investigators from different centers were connected via GeneMatcher. Most individuals had developmental delay and generalized epilepsy with onset in the first 2 years. However, we found a broad spectrum of phenotypic severity, ranging from early normal development with pharmacoresponsive seizures to profound intellectual disability with intractable epilepsy and recurrent refractory status epilepticus. These findings suggest that CSNK2B should be considered in the diagnostic evaluation of patients with a broad range of NDD with treatable or intractable seizures.

Purpose Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. Methods From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. Results The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. Conclusion Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

BackgroundDystonia is a clinically and genetically heterogeneous movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements and/or postures. Heterozygous variants in lysine methyltransferase 2B (KMT2B), encoding a histone H3 methyltransferase, have been associated with a childhood-onset, progressive and complex form of dystonia (dystonia 28, DYT28). Since 2016, more than one hundred rareKMT2Bvariants have been reported, including frameshift, nonsense, splice site, missense and other in-frame changes, many having an uncertain clinical impact.ResultsWe characterize the genome-wide peripheral blood DNA methylation profiles of a cohort of 18 patients with pathogenic and unclassifiedKMT2Bvariants. We resolve the “episignature” associated withKMT2Bhaploinsufficiency, proving that this approach is robust in diagnosing clinically unsolved cases, properly classifying them with respect to other partially overlapping dystonic phenotypes, other rare neurodevelopmental disorders and healthy controls. Notably, defective KMT2B function in DYT28 causes a non-random DNA hypermethylation across the genome, selectively involving promoters and other regulatory regions positively controlling gene expression.ConclusionsWe demonstrate a distinctive DNA hypermethylation pattern associated with DYT28, provide an epigenetic signature for this disorder enabling accurate diagnosis and reclassification of ambiguous genetic findings and suggest potential therapeutic approaches.

SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system. SPTBN1 mutations cause a neurodevelopmental syndrome characterized by intellectual disability, language and motor delays, autism, seizures and other features. The variants disrupt βII-spectrin function and disturb cytoskeletal organization and dynamics.

Contains fulltext : 234078.pdf (Publisher’s version ) (Open Access) Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Contains fulltext : 231702.pdf (Publisher’s version ) (Closed access) Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy., German Research Foundation (DFG); European Union (European Union); NEUROMICS Network; International Coordination Action (ICA); Fund for Scientific Research Flanders (FWO); Netherlands Organization for Scientific Research (ZONMW VIDI); National Medical Research Council, Singapore; A Strategic Positioning Fund on Genetic Orphan Diseases (GODAFIT); Industry Alignment Fund on Singapore Childhood Undiagnosed Diseases Program (SUREKids); Biomedical Research Council, A*STAR; Diana and Steve Marienhoff Fashion Industries Guild Endowed Fellowship in Pediatric Neuromuscular Diseases; Fondazione Bambino Gesù (Vite Coraggiose); Canadian Institutes of Health Research; Natural Sciences and Engineering Research Council of Canada; Eurocores Program EuroEPINOMICS; University of Antwerp Research Fund; FRAXA Foundation; Brain & Behavior Research Foundation, NARSAD Young Investigator Grant

SPTBN1 encodes βII-spectrin, the ubiquitously expressed member of the β-spectrin family that forms micrometer-scale networks associated with plasma membranes. βII-spectrin is abundantly expressed in the brain, where it is essential for neuronal development and connectivity. Mice deficient in neuronal βII-spectrin expression have defects in cortical organization, global developmental delay, dysmorphisms, and behavioral deficiencies of corresponding severity. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous variants in this gene may also present with measurable compromise of neural development and function. Here we report the identification of heterozygous SPTBN1 variants in 29 individuals who present with global developmental, language and motor delays, mild to severe intellectual disability, autistic features, seizures, behavioral and movement abnormalities, hypotonia, and variable dysmorphic facial features. We show that these SPTBN1 variants lead to loss-of-function, gain-of-function, and dominant negative effects that affect protein stability, disrupt binding to key protein partners, and disturb cytoskeleton organization and dynamics. Our studies define the genetic basis of this new neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and neural development, and underscore the critical role of βII-spectrin in the central nervous system.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

Mitochondrial disorders are caused by nuclear and mitochondrial pathogenic variants leading to defects in mitochondrial function and cellular respiration. Recently, the nuclear-encoded mitochondrial fusion gene MSTO1 (Misato 1) has been implicated in mitochondrial myopathy and ataxia. Here we report on a 30-yr-old man presenting with a maternally inherited NM_018116.3:c.651C>G, p.F217L missense variant as well as a paternally inherited arr[GRCh37] 1q22(155581773_155706887) × 1 deletion encompassing exons 7–14 of MSTO1. His phenotype included muscle weakness, hypotonia, early motor developmental delay, pectus excavatum, and scoliosis. Testing revealed elevated plasma creatine kinase, and electromyogram results were consistent with longstanding generalized myopathy. These phenotypic features overlap well with previously reported patients harboring biallelic MSTO1 variants. Additionally, our patient presents with dysphagia and restrictive lung disease, not previously reported for MSTO1-associated disorders. The majority of patients with disease-associated variants in MSTO1 present with biallelic variants suggesting autosomal recessive inheritance; however, one family has been reported with a single variant and presumed autosomal dominant inheritance. The pattern of inheritance we observed is consistent with the majority of previous reports suggesting an autosomal recessive disorder. We add to our knowledge of the syndrome caused by variants in MSTO1 and provide additional evidence supporting autosomal recessive inheritance. We also describe phenotypic features not reported in previous cases, although further research is needed to confirm they are associated with defects in MSTO1.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.

Mitochondrial disorders are caused by nuclear and mitochondrial pathogenic variants leading to defects in mitochondrial function and cellular respiration. Recently, the nuclear-encoded mitochondrial fusion gene MSTO1 (Misato 1) has been implicated in mitochondrial myopathy and ataxia. Here we report on a 30-yr-old man presenting with a maternally inherited NM_018116.3:c.651C>G, p.F217L missense variant as well as a paternally inherited arr[GRCh37] 1q22(155581773_155706887) × 1 deletion encompassing exons 7–14 of MSTO1. His phenotype included muscle weakness, hypotonia, early motor developmental delay, pectus excavatum, and scoliosis. Testing revealed elevated plasma creatine kinase, and electromyogram results were consistent with longstanding generalized myopathy. These phenotypic features overlap well with previously reported patients harboring biallelic MSTO1 variants. Additionally, our patient presents with dysphagia and restrictive lung disease, not previously reported for MSTO1-associated disorders. The majority of patients with disease-associated variants in MSTO1 present with biallelic variants suggesting autosomal recessive inheritance; however, one family has been reported with a single variant and presumed autosomal dominant inheritance. The pattern of inheritance we observed is consistent with the majority of previous reports suggesting an autosomal recessive disorder. We add to our knowledge of the syndrome caused by variants in MSTO1 and provide additional evidence supporting autosomal recessive inheritance. We also describe phenotypic features not reported in previous cases, although further research is needed to confirm they are associated with defects in MSTO1.

BackgroundRNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data.MethodsWe describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization.ResultsWe demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance.ConclusionsThe approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.

Diagnostic exome sequencing yields a single genetic diagnosis in ∼30% of cases, and according to recent studies the prevalence of identifying two genetic conditions in a single individual range between 4.6% and 7%. We present a patient diagnosed with three different rare conditions, each explained by a pathogenic variant in a different gene. A 17-yr-old female was evaluated for a history of motor and speech delay, scoliosis, distinctive craniofacial features, and dry skin in the Department of Clinical Genomics at Mayo Clinic. Her distinctive features included prominent forehead, epicanthus, depressed nasal bridge, narrow mouth, prognathism, malar flattening, and oligodontia. Family history was notable for dry skin in her mother and missing teeth in the paternal grandmother. Previous diagnostic testing was unrevealing including biochemical testing, echocardiogram, abdominal ultrasound, and electroencephalogram. Previous genetic testing included a microarray-based comparative genomic hybridization that was reported normal. Three pathogenic loss-of-function heterozygous variants were identified by exome trio sequencing, each linked to different genetic conditions: SIN3A (Witteveen–Kolk syndrome), FLG (dermatitis), and EDAR (ectodermal dysplasia). Together, these three genetic alterations could explain the patient's overall phenotype. This patient demonstrates the importance of performing a thorough curation of exome data when presented with a complex phenotype. Although phenotypic variability can explain some of these situations, the hypothesis of multiple diseases coexisting in a single patient should never be disregarded completely.