pinecone/core-2020-05-10-deduplication huggingface dataset:
Functional studies of tyrosine hydroxylase missense variants reveal distinct patterns of molecular defects in dopa-responsive dystonia

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients
Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS
Impaired Function is a Common Feature of Neuropathy‐Associated Glycyl‐t RNA  Synthetase Mutations

C harcot– M arie– T ooth disease type 2 D  ( CMT 2 D ) is an autosomal‐dominant axonal peripheral neuropathy characterized by impaired motor and sensory function in the distal extremities. Mutations in the glycyl‐t RNA  synthetase (  GARS  ) gene cause  CMT 2 D .  GARS  is a member of the ubiquitously expressed aminoacyl‐ tRNA  synthetase ( ARS ) family and is responsible for charging t RNA  with glycine. To date, 13   GARS   mutations have been identified in patients with  CMT  disease. While functional studies have revealed loss‐of‐function characteristics, only four   GARS   mutations have been rigorously studied. Here, we report the functional evaluation of nine  CMT ‐associated   GARS   mutations in t RNA  charging, yeast complementation, and subcellular localization assays. Our results demonstrate that impaired function is a common characteristic of  CMT ‐associated   GARS   mutations. Additionally, one mutation previously associated with  CMT  disease (p. S er581 L eu) does not demonstrate impaired function, was identified in the general population, and failed to segregate with disease in two newly identified families with  CMT  disease. Thus, we propose that this variant is not a disease‐causing mutation. Together, our data indicate that impaired function is a key component of   GARS  ‐mediated  CMT  disease and emphasize the need for careful genetic and functional evaluation before implicating a variant in disease onset
Nonsense Mutation in Coiled-Coil Domain Containing 151 Gene (CCDC151) Causes Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder characterized by impaired ciliary function that leads to subsequent clinical phenotypes such as chronic sinopulmonary disease. PCD is also a genetically heterogeneous disorder with many single gene mutations leading to similar clinical phenotypes. Here, we present a novel PCD causal gene, coiled-coil domain containing 151 (CCDC151), which has been shown to be essential in motile cilia of many animals and other vertebrates but its effects in humans was not observed until currently. We observed a novel nonsense mutation in a homozygous state in the CCDC151 gene (NM_145045.4:c.925G>T:p.[E309*]) in a clinically diagnosed PCD patient from a consanguineous family of Arabic ancestry. The variant was absent in 238 randomly selected individuals indicating that the variant is rare and likely not to be a founder mutation. Our finding also shows that given prior knowledge from model organisms, even a single whole-exome sequence can be sufficient to discover a novel causal gene.Peer-reviewedPublisher Versio
Variants in CUL4B are associated with cerebral malformations

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B
Functional classification of BRCA2 DNA variants by splicing assays in a large minigene with 9 exons

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License.Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19 to 27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations.Contract grant sponsors: Instituto de Salud Carlos III (Spanish Ministry of Economy and Competitiveness) grants PI10/2910 and PI13/1749, Consejería de Sanidad (Junta de Castilla y León) grant BIO/VA08/13. AA was supported by the European Social Fund and Consejeria de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007–2013. CHM is supported by Fundación Villalar (Castilla y León, Spain).Peer reviewe
De novo heterozygous mutations in SMC3 cause a range of cornelia de lange syndrome-overlapping phenotypes

© 2015 WILEY PERIODICALS, INC. Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ~1%-2% of CdLS-like phenotypes. Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder caused by mutation in five genes encoding subunits or regulators of the cohesin complex. To date, only the clinical features of the unique mildly affected CdLS male with SMC3 mutation have been published. Here, we report a series of 16 probands with 15 different intragenic mutations in SMC3 that provide a significant advance in our understanding of the clinical and molecular basis of Cornelia de Lange syndrome and overlapping phenotypes.CdLS Foundation of UK and Ireland for their long-term help and support. M.A.D. and I.D.K. are indebted to the USA Cornelia de Lange Syndrome FoundationPeer Reviewe
Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real-time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in 7 unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits thereby interfering with integrin maturation and/or function. Our study extends knowledge of Glanzmann thrombasthenia and the pathophysiology of an integrin. This article is protected by copyright. All rights reserved
Understanding the implications of mitochondrial DNA variation in the health of black Southern African populations : the 2014 workshop

Knowledge concerning the prevalence and severity of disease
 related to/associated with variations in mitochondrial DNA
 (mtDNA) in Black African populations lags behind that of other
 groups from a global perspective. Developing an understanding of
 the variants that cause mitochondrial disease in this population is
 clearly interesting from a scientific perspective, and is also of
 importance to health services in Africa and nations outside of the
 African continent with appreciable populations of African origin.
 In addition to identifying mtDNA mutations causing
 mitochondrial disease, unravelling the role of mtDNA variants in
 complex disease is of great relevance and will require the coming
 together of many emerging specialities in southern Africa. This
 effort will be aided if conducted in a wider collaborative manner in
 the context of forums such as the South African Human Genome
 Programme (SAHGP; http://sahgp.sanbi.ac.za), H3 Africa, and the
 Human Variome Project (HVP; http://
 www.humanvariomeproject.org).Royal Society and theNationalResearch Foundation of SouthAfrica.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1098-10042016-05-31hb201
Molecular diversity and associated phenotypic spectrum of germline CBL mutations

Noonan syndrome (NS) is a relatively common developmental disorder with a pleomorphic phenotype. Mutations causing NS alter genes encoding proteins involved in the RAS-MAPK pathway. We and others identified Casitas B-lineage lymphoma proto-oncogene (CBL), which encodes an E3-ubiquitin ligase acting as a tumor suppressor in myeloid malignancies, as a disease gene underlying a condition clinically related to NS. Here, we further explored the spectrum of germline CBL mutations and their associated phenotype. CBL mutation scanning performed on 349 affected subjects with features overlapping NS and no mutation in NS genes allowed the identification of five different variants with pathological significance. Among them, two splice-site changes, one in-frame deletion, and one missense mutation affected the RING domain and/or the adjacent linker region, overlapping cancer-associated defects. A novel nonsense mutation generating a v-Cbl-like protein able to enhance signal flow through RAS was also identified. Genotype-phenotype correlation analysis performed on available records indicated that germline CBL mutations cause a variable phenotype characterized by a relatively high frequency of neurological features, predisposition to juvenile myelomonocytic leukemia, and low prevalence of cardiac defects, reduced growth, and cryptorchidism. Finally, we excluded a major contribution of two additional members of the CBL family, CBLB and CBLC, to NS and related disorders
The Effects of PMM2-CDG-Causing Mutations on the Folding, Activity, and Stability of the PMM2 Protein

© 2015 WILEY PERIODICALS, INC.. Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The results suggest that PMM2-CDG could be considered a conformational disease and therefore PMM2 activity could be rescuable via the use of small stabilizer molecules. Unfortunately, this mouse model is inadequate for testing that kind of compounds since neither mutation causes conformational instability.Peer Reviewe
KIAA0586 is mutated in Joubert syndrome

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive mid-hindbrain malformation. JS is part of a group of disorders called ciliopathies based on their overlapping phenotypes and common underlying pathophysiology linked to primary cilium dysfunction. Biallelic mutations in one of 28 genes, all encoding proteins localizing to the primary cilium or basal body, can cause JS. Despite this large number of genes, the genetic cause can currently be determined in about 62% of individuals with JS. To identify novel JS genes, we performed whole exome sequencing on 35 individuals with JS and found biallelic rare deleterious variants (RDVs) in KIAA0586, encoding a centrosomal protein required for ciliogenesis, in one individual. Targeted next-generation sequencing in a large JS cohort identified biallelic RDVs in eight additional families for an estimated prevalence of 2.5% (9/366 JS families). All affected individuals displayed JS phenotypes toward the mild end of the spectrum
High incidence of Noonan syndrome features including short stature and pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P<0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients
Hidden genetic variation in LCA9-associated congenital blindness explained by 5′UTR mutations and copy-number variations of NMNAT1

Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness
Targeted Next Generation Sequencing Analysis of 1000 individuals with Intellectual Disability

To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ∼11% of the cases (113 variants in 107/986 individuals: ∼8% of the individuals had a likely pathogenic loss of function (LoF) variant, while ∼3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2 and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modelling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10-15% yield from array CGH alone.Action Medical Research (SP4640); the Birth Defect Foundation (RG45448); the Cambridge National Institute for Health Research Biomedical Research Centre (RG64219); the NIHR Rare Diseases BioResource (RBAG163); Wellcome Trust award WT091310; The Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation and other genetic diseases (member of the Telethon Network of Genetic Biobanks (project no. GTB12001); the Genetic Origins of Congenital Heart Disease Study (GO-CHD)- funded by British Heart Foundation (BHF)This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/humu.2290