RESUMEN
PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.
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Displasia Ectodérmica , Factor de Unión 1 al Potenciador Linfoide/genética , Vía de Señalización Wnt , Consanguinidad , Displasia Ectodérmica/genética , Humanos , Deformidades Congénitas de las Extremidades , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Síndrome , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Essential tremor (ET) is a neurological disorder characterized by bilateral and symmetric postural, isometric, and kinetic tremors of forelimbs produced during voluntary movements. To date, only a single SCN4A variant has been suggested to cause ET. In continuation of the previous report on the association between SCN4A and ET in a family from Spain, we validated the pathogenicity of a novel SCN4A variant and its involvement in ET in a second family affected by this disease. We recruited a Kurdish family with four affected members manifesting congenital tremor. Using whole-exome sequencing, we identified a novel missense variant in SCN4A, NM_000334.4:c.4679C>T; p.(Pro1560Leu), thus corroborating SCN4A's role in ET. The residue is highly conserved across vertebrates and the substitution is predicted to be pathogenic by various in silico tools. Western blotting and immunocytochemistry performed in cells derived from one of the patients showed reduced immunoreactivity of SCN4A as compared to control cells. The study provides supportive evidence for the role of SCN4A in the etiology of ET and expands the phenotypic spectrum of channelopathies to this neurological disorder.
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Canalopatías , Temblor Esencial , Animales , Consanguinidad , Temblor Esencial/genética , Humanos , Mutación Missense/genética , Canal de Sodio Activado por Voltaje NAV1.4/genética , LinajeRESUMEN
Autism spectrum disorder (ASD) is a group of neurological and developmental disabilities characterised by clinical and genetic heterogeneity. The current study aimed to expand ASD genotyping by investigating potential associations with SYNE2 mutations. Specifically, the disease-causing variants of SYNE2 in 410 trios manifesting neurodevelopmental disorders using whole-exome sequencing were explored. The consequences of the identified variants were studied at the transcript level using quantitative polymerase chain reaction (qPCR). For validation, immunofluorescence and immunoblotting were performed to analyse mutational effects at the protein level. The compound heterozygous variants of SYNE2 (NM_182914.3:c.2483T>G; p.(Val828Gly) and NM_182914.3:c.2362G>A; p.(Glu788Lys)) were identified in a 4.5-year-old male, clinically diagnosed with autism spectrum disorder, developmental delay and intellectual disability. Both variants reside within the nesprin-2 giant spectrin repeat (SR5) domain and are predicted to be highly damaging using in silico tools. Specifically, a significant reduction of nesprin-2 giant protein levels is revealed in patient cells. SYNE2 transcription and the nuclear envelope localisation of the mutant proteins was however unaffected as compared to parental control cells. Collectively, these data provide novel insights into the cardinal role of the nesprin-2 giant in neurodevelopment and suggest that the biallelic hypomorphic SYNE2 mutations may be a new cause of intellectual disability and ASD.
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Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Células Cultivadas , Niño , Heterocigoto , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Mutación Missense , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Dominios Proteicos , Transporte de ProteínasRESUMEN
PURPOSE: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition. METHODS: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable. RESULTS: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca2+-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4. CONCLUSION: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans.
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Discapacidad Intelectual , Microcefalia , Trastornos del Neurodesarrollo , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Humanos , Discapacidad Intelectual/genética , Microcefalia/genética , Trastornos del Neurodesarrollo/genética , Linaje , Fenotipo , Convulsiones/genéticaRESUMEN
Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH (n = 3) or Seckel syndrome (n = 2). In addition to homozygous causal variants in ASPM or CENPJ, we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT-genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.
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Genes Modificadores , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Ácido Anhídrido Hidrolasas/genética , Adulto , Antígenos/genética , Proteínas de Ciclo Celular/genética , Niño , Proteínas de Unión al ADN/genética , Femenino , Heterocigoto , Humanos , Masculino , Microcefalia/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Linaje , FenotipoRESUMEN
Leukodystrophies are genetic disorders of cerebral white matter that almost exclusively have a progressive disease course. We became aware of three members of a family with a disorder characterized by a sudden loss of all previously acquired abilities around 1 year of age followed by almost complete recovery within 2 years. Cerebral MRI and myelin sensitive imaging showed a pronounced demyelination that progressed for several months despite signs of clinical improvement and was followed by remyelination. Exome sequencing did not-identify any mutations in known leukodystrophy genes but revealed a heterozygous variant in the FBP2 gene, c.343G>A, p. Val115Met, shared by the affected family members. Cerebral MRI of other family members demonstrated similar white matter abnormalities in all carriers of the variant in FBP2. The FBP2 gene codes for muscle fructose 1,6-bisphosphatase, an enzyme involved in gluconeogenesis that is highly expressed in brain tissue. Biochemical analysis showed that the variant has a dominant negative effect on enzymatic activity, substrate affinity, cooperativity and thermal stability. Moreover, it also affects the non-canonical functions of muscle fructose 1,6-bisphosphatase involved in mitochondrial protection and regulation of several nuclear processes. In patients' fibroblasts, muscle fructose 1,6-bisphosphatase shows no colocalization with mitochondria and nuclei leading to increased reactive oxygen species production and a disturbed mitochondrial network. In conclusion, the results of this study indicate that the variant in FBP2 disturbs cerebral energy metabolism and is associated with a novel remitting leukodystrophy.
RESUMEN
OBJECTIVE: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality. METHODS: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model. RESULTS: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied. INTERPRETATION: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.
Asunto(s)
Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad/genética , Proteínas Mitocondriales/genética , Atrofia Óptica Autosómica Dominante/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Ligamiento Genético/genética , Humanos , Masculino , Ratones , Mutación Missense , Atrofia Óptica Autosómica Dominante/patología , Linaje , ARN Mensajero/genética , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Pez Cebra/genéticaRESUMEN
OBJECTIVE: Autosomal recessive primary microcephaly (MCPH) is a rare condition characterized by a reduced cerebral cortex accompanied with intellectual disability. Mutations in 17 genes have been shown to cause this phenotype. Recently, mutations in CIT, encoding CRIK (citron rho-interacting kinase)-a component of the central spindle matrix-were added. We aimed at identifying novel MCPH-associated genes and exploring their functional role in pathogenesis. METHODS: Linkage analysis and whole exome sequencing were performed in consanguineous and nonconsanguineous MCPH families to identify disease-causing variants. Functional consequences were investigated by RNA studies and on the cellular level using immunofluorescence and microscopy. RESULTS: We identified homozygous mutations in KIF14 (NM_014875.2;c.263T>A;pLeu88*, c.2480_2482delTTG; p.Val827del, and c.4071G>A;p.Gln1357=) as the likely cause in 3 MCPH families. Furthermore, in a patient presenting with a severe form of primary microcephaly and short stature, we identified compound heterozygous missense mutations in KIF14 (NM_014875.2;c.2545C>G;p.His849Asp and c.3662G>T;p.Gly1221Val). Three of the 5 identified mutations impaired splicing, and 2 resulted in a truncated protein. Intriguingly, Kif14 knockout mice also showed primary microcephaly. Human kinesin-like protein KIF14, a microtubule motor protein, localizes at the midbody to finalize cytokinesis by interacting with CRIK. We found impaired localization of both KIF14 and CRIK at the midbody in patient-derived fibroblasts. Furthermore, we observed a large number of binucleated and apoptotic cells-signs of failed cytokinesis that we also observed in experimentally KIF14-depleted cells. INTERPRETATION: Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577.
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Citocinesis/genética , Regulación de la Expresión Génica/genética , Cinesinas/genética , Microcefalia/genética , Mutación/genética , Proteínas Oncogénicas/genética , Caspasa 7/metabolismo , Movimiento Celular/genética , Células Cultivadas , Niño , Preescolar , Salud de la Familia , Femenino , Fibroblastos/fisiología , Estudio de Asociación del Genoma Completo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Microcefalia/diagnóstico por imagen , Microcefalia/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.
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Alelos , Autoantígenos/genética , Deformidades Congénitas de la Mano/complicaciones , Deformidades Congénitas de la Mano/genética , Hiperhidrosis/complicaciones , Hiperhidrosis/genética , Mutación , Retinitis Pigmentosa/complicaciones , Retinitis Pigmentosa/genética , Trismo/congénito , Secuencia de Aminoácidos , Autoantígenos/química , Niño , Preescolar , Muerte Súbita , Facies , Femenino , Humanos , Lactante , Masculino , Modelos Moleculares , Linaje , Fenotipo , Síndrome , Trismo/complicaciones , Trismo/genéticaRESUMEN
Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E-NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PPi ), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal-recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PPi generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild-type E-NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E-NPP1 enzyme activity were still able to generate PPi and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E-NPP1 function, which would be potential therapeutical targets for conformational-stabilizing agents.
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Mutación Missense , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Calcificación Vascular/genética , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Difosfatos/metabolismo , Regulación hacia Abajo , Células HEK293 , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
We describe a large family with disproportionate short stature and bone dysplasia from Nias in which we observed differences in severity when comparing the phenotypes of affected individuals from two remote branches. We conducted a linkage scan in the more severely affected family branch and determined a critical interval of 4.7 cM on chromosome 11. Sequencing of the primary candidate gene TBX10 did not reveal a disease-causing variant. When performing whole exome sequencing we noticed a homozygous missense variant in B3GAT3, c.419C>T [p.(Pro140Leu)]. B3GAT3 encodes ß-1,3-glucuronyltransferase-I (GlcAT-I). GlcAT-I catalyzes an initial step of proteoglycan synthesis and the mutation p. (Pro140Leu) lies within the donor substrate-binding subdomain of the catalytic domain. In contrast to the previously published mutation in B3GAT3, c.830G>A [p.(Arg277Gln)], no heart phenotype could be detected in our family. Functional studies revealed a markedly reduced GlcAT-I activity in lymphoblastoid cells from patients when compared to matched controls. Moreover, relative numbers of glycosaminoglycan (GAG) side chains were decreased in patient cells. We found that Pro140Leu-mutant GlcAT-I cannot efficiently transfer GlcA to the linker region trisaccharide. This failure results in a partial deficiency of both chondroitin sulfate and heparan sulfate chains. Since the phenotype of the Nias patients differs from the Larsen-like syndrome described for patients with mutation p.(Arg277Gln), we suggest mutation B3GAT3:p.(Pro140Leu) to cause a different type of GAG linkeropathy showing no involvement of the heart.
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Enfermedades del Desarrollo Óseo/genética , Enfermedades Genéticas Congénitas/genética , Mutación Missense , Adolescente , Adulto , Sustitución de Aminoácidos , Enfermedades del Desarrollo Óseo/enzimología , Enfermedades del Desarrollo Óseo/patología , Niño , Preescolar , Femenino , Enfermedades Genéticas Congénitas/enzimología , Enfermedades Genéticas Congénitas/patología , Glucuronosiltransferasa/metabolismo , Humanos , Lactante , Masculino , Linaje , Estructura Terciaria de ProteínaRESUMEN
Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals' blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption.
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Enfermedades de la Aorta/genética , ARN Helicasas DEAD-box/genética , Hipoplasia del Esmalte Dental/genética , Metacarpo/anomalías , Modelos Moleculares , Enfermedades Musculares/genética , Odontodisplasia/genética , Osteoporosis/genética , Fenotipo , Calcificación Vascular/genética , Secuencia de Aminoácidos , Arterias/patología , Secuencia de Bases , Calcinosis/genética , Calcinosis/patología , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Exoma/genética , Genes Dominantes/genética , Humanos , Inmunohistoquímica , Helicasa Inducida por Interferón IFIH1 , Interferón beta/metabolismo , Datos de Secuencia Molecular , Mutación Missense/genética , Linaje , Análisis de Secuencia de ADN , Anomalías Dentarias/genética , Anomalías Dentarias/patologíaRESUMEN
Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.
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Trastornos del Crecimiento/genética , Microcefalia/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Degeneración Retiniana/genética , Adolescente , Adulto , Animales , Centriolos/ultraestructura , Niño , Preescolar , Salud de la Familia , Femenino , Fibroblastos/metabolismo , Genotipo , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Repeticiones de Microsatélite , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Pakistán , Linaje , Fenotipo , Adulto Joven , Pez CebraRESUMEN
Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.
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Pueblo Asiatico/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Mutación Missense , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Niño , Preescolar , Exoma , Femenino , Humanos , Masculino , Microcefalia/genética , Microcefalia/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Pakistán , Linaje , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.
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Centrosoma/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Mitosis/genética , Mapeo Cromosómico , Cromosomas Humanos Par 7/genética , Quinasa 6 Dependiente de la Ciclina/química , Quinasa 6 Dependiente de la Ciclina/metabolismo , Femenino , Estudios de Asociación Genética , Humanos , Discapacidad Intelectual/fisiopatología , Masculino , Microcefalia/fisiopatología , Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Conformación ProteicaRESUMEN
We report on a 25-year-old female with intellectual disability, mildly unusual face, and a pervasive developmental disorder, in whom routine aCGH showed a 298 kb de novo deletion at chromosome 2q24.1(156869529-157167986 × 1). The region contained two genes (NR4A2; GPD2). Molecular studies in the proposita showed an additional variant in GPD2 (c.614C > T, p.Pro205Leu), which was predicted to be pathogenic. The variant was also present in the healthy mother and sister. Functional analysis showed absent GPD2 activity in the proposita and 50% activity in mother and sister. We conclude that we have been able to find circumstantial evidence for the causative effect of the hemizygous GPD2 mutation but full proof remained lacking. Total costs for the work-up in these patients were high (21,975 [$27,029]). Similar results will increasingly be found when Next Generation Techniques will be applied widely in patients with intellectual disability, and proving pathogenicity by functional studies or in animal models will be expensive. We advocate the use of freely accessible international databases combining phenotype and genotype data using standard nomenclatures to facilitate proving pathogenicity of research data and to decrease costs of health care.
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Glicerolfosfato Deshidrogenasa/genética , Hemicigoto , Discapacidad Intelectual/genética , Adulto , Hibridación Genómica Comparativa , Femenino , Humanos , Masculino , Mutación , Análisis de Secuencia de ADN/economíaRESUMEN
Inherited disorders of vitamin B12 (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B12 from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B12.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Anomalías Múltiples/genética , Errores Innatos del Metabolismo/genética , Mutación , Vitamina B 12/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Anomalías Múltiples/enzimología , Estudios de Casos y Controles , Células Cultivadas , Análisis Mutacional de ADN , Fibroblastos/metabolismo , Expresión Génica , Genes Recesivos , Estudios de Asociación Genética , Humanos , Recién Nacido , Proteínas de Membrana de los Lisosomas/metabolismo , Errores Innatos del Metabolismo/enzimología , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación , Hidrolasas Diéster Fosfóricas/genética , Seudoxantoma Elástico/genética , Pirofosfatasas/genética , Calcificación Vascular/genética , Estrías Angioides/genética , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Seudoxantoma Elástico/patología , Estudios Retrospectivos , Encuestas y Cuestionarios , Calcificación Vascular/patologíaRESUMEN
Glutathione peroxidases (GPx) form a heterogeneous enzyme family and GPx4-isoforms have been implicated in anti-oxidative defense, brain development, neuroinjury and sperm maturation. In humans seven GPx isoforms (GPx1-GPx7) can be separated. To selectively quantify the expression of GPx4-isoforms we have raised a monoclonal antibody (anti-hGPx4 Mab63-1) against the pure recombinant Sec46Cys mutant of human cytosolic GPx4 and used it for immunoblotting, immunoprecipitation and immunohistochemistry. The antibody recognizes human GPx4, its mouse ortholog but neither reacted with rat GPx4 nor other human GPx-isoforms. Sequence alignment of human and rat GPx4 proteins indicated three different amino acids (S18, F35, K99 in humans, A18, C35, R99 in rats) and a S18A exchange in the human enzyme completely abolished immunoreactivity. To further characterize the immunological epitope we synthesized a set of 12-mer peptides flanking S18* of human GPx4 and found that the sequence SMHEFS*AKDIDG exhibited strongest immunoreactivity. Substitution analysis and peptide length variation narrowed down the essential epitope to FS*AKDI and indicated that most mutations in this region strongly impaired immunoreactivity. In silico blast searches of public protein databases failed to identify proteins with potential immunoreactivity suggesting that the antibody exhibits a high specificity for human and mouse GPx4 and may not cross-react with unrelated proteins.