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1.
Hum Mutat ; 40(8): 1063-1070, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31045292

RESUMO

Microcephalic primordial dwarfism (MPD) is a group of rare single-gene disorders characterized by the extreme reduction in brain and body size from early development onwards. Proteins encoded by MPD-associated genes play important roles in fundamental cellular processes, notably genome replication and repair. Here we report the identification of four MPD individuals with biallelic variants in DNA2, which encodes an adenosine triphosphate (ATP)-dependent helicase/nuclease involved in DNA replication and repair. We demonstrate that the two intronic variants (c.1764-38_1764-37ins(53) and c.74+4A>C) found in these individuals substantially impair DNA2 transcript splicing. Additionally, we identify a missense variant (c.1963A>G), affecting a residue of the ATP-dependent helicase domain that is highly conserved between humans and yeast, with the resulting substitution (p.Thr655Ala) predicted to directly impact ATP/ADP (adenosine diphosphate) binding by DNA2. Our findings support the pathogenicity of these variants as biallelic hypomorphic mutations, establishing DNA2 as an MPD disease gene.

2.
Am J Hum Genet ; 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30503519

RESUMO

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

3.
Nat Genet ; 2018 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478443

RESUMO

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.

4.
Am J Hum Genet ; 103(4): 553-567, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30290151

RESUMO

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

6.
Am J Hum Genet ; 103(2): 221-231, 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30057030

RESUMO

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

7.
Am J Med Genet A ; 176(2): 465-469, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29265708

RESUMO

RNU4ATAC pathogenic variants to date have been associated with microcephalic osteodysplastic primordial dwarfism, type 1 and Roifman syndrome. Both conditions are clinically distinct skeletal dysplasias with microcephalic osteodysplastic primordial dwarfism, type 1 having a more severe phenotype than Roifman syndrome. Some of the overlapping features of the two conditions include developmental delay, microcephaly, and immune deficiency. The features also overlap with Lowry Wood syndrome, another rare but well-defined skeletal dysplasia for which the genetic etiology has not been identified. Characteristic features include multiple epiphyseal dysplasia and microcephaly. Here, we describe three patients with Lowry Wood syndrome with biallelic RNU4ATAC pathogenic variants. This report expands the phenotypic spectrum for biallelic RNU4ATAC disorder causing variants and is the first to establish the genetic cause for Lowry Wood syndrome.

8.
Am J Hum Genet ; 101(5): 856-865, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100095

RESUMO

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.


Assuntos
Aciltransferases/genética , Atrofia/genética , Doenças Ósseas Metabólicas/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Adolescente , Adulto , Alelos , Cerebelo/patologia , Criança , Pré-Escolar , Exoma/genética , Feminino , Fibroblastos/patologia , Glicosilfosfatidilinositóis/genética , Humanos , Masculino , Hipotonia Muscular/genética , Linhagem , RNA Mensageiro/genética , Convulsões/genética
9.
Nat Genet ; 49(4): 537-549, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28191891

RESUMO

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.


Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Nanismo/genética , Instabilidade Genômica/genética , Microcefalia/genética , Mutação/genética , Linhagem Celular , Dano ao DNA/genética , Feminino , Humanos , Masculino
10.
Genome Biol ; 17(1): 242, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27894351

RESUMO

BACKGROUND: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete. RESULTS: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population. CONCLUSIONS: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.


Assuntos
Cílios/genética , Transtornos da Motilidade Ciliar/genética , Ciliopatias/genética , Encefalocele/genética , Mutação/genética , Doenças Renais Policísticas/genética , Alelos , Cílios/patologia , Transtornos da Motilidade Ciliar/patologia , Ciliopatias/patologia , Análise Mutacional de DNA , Encefalocele/patologia , Estudos de Associação Genética , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Fenótipo , Doenças Renais Policísticas/patologia , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa
11.
Am J Hum Genet ; 99(4): 984-990, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27693231

RESUMO

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.


Assuntos
Amelogênese Imperfeita/genética , Mutação , Receptores Acoplados a Proteínas-G/genética , Amelogênese/genética , Animais , Sequência de Bases , Esmalte Dentário/crescimento & desenvolvimento , Esmalte Dentário/patologia , Feminino , Homozigoto , Humanos , Concentração de Íons de Hidrogênio , Masculino , Linhagem , Ratos , Receptores Acoplados a Proteínas-G/análise
12.
Am J Hum Genet ; 98(4): 615-26, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26996948

RESUMO

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 various proteins to the cell surface. At least 27 genes are involved in biosynthesis and transport of GPI-anchored proteins (GPI-APs). To date, mutations in 13 of these genes are known to cause inherited GPI deficiencies (IGDs), and all are inherited as recessive traits. IGDs mainly manifest as intellectual disability, epilepsy, coarse facial features, and multiple organ anomalies. These symptoms are caused by the decreased surface expression of GPI-APs or by structural abnormalities of GPI. Here, we present five affected individuals (from two consanguineous families from Egypt and Pakistan and one non-consanguineous family from Japan) who show intellectual disability, hypotonia, and early-onset seizures. We identified pathogenic variants in PIGG, a gene in the GPI pathway. In the consanguineous families, homozygous variants c.928C>T (p.Gln310(∗)) and c.2261+1G>C were found, whereas the Japanese individual was compound heterozygous for c.2005C>T (p.Arg669Cys) and a 2.4 Mb deletion involving PIGG. PIGG is the enzyme that modifies the second mannose with ethanolamine phosphate, which is removed soon after GPI is attached to the protein. Physiological significance of this transient modification has been unclear. Using B lymphoblasts from affected individuals of the Egyptian and Japanese families, we revealed that PIGG activity was almost completely abolished; however, the GPI-APs had normal surface levels and normal structure, indicating that the pathogenesis of PIGG deficiency is not yet fully understood. The discovery of pathogenic variants in PIGG expands the spectrum of IGDs and further enhances our understanding of this etiopathogenic class of intellectual disability.


Assuntos
Variação Genética , Glicosilfosfatidilinositóis/genética , Deficiência Intelectual/genética , Manosiltransferases/genética , Hipotonia Muscular/genética , Convulsões/genética , Anormalidades Múltiplas/genética , Adolescente , Linhagem Celular Tumoral , Criança , Consanguinidade , Egito , Técnicas de Genotipagem , Glicosilfosfatidilinositóis/metabolismo , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Lactente , Japão , Mutação , Paquistão , Linhagem , Adulto Jovem
14.
Am J Hum Genet ; 97(6): 878-85, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26626625

RESUMO

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.


Assuntos
Colágeno Tipo XIII/genética , Mutação , Síndromes Miastênicas Congênitas/genética , Mioblastos/metabolismo , Junção Neuromuscular/metabolismo , Adulto , Animais , Linhagem Celular , Pré-Escolar , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Colágeno Tipo XIII/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Exoma , Feminino , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Camundongos , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/patologia , Mioblastos/patologia , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/patologia , Linhagem , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapses/genética , Sinapses/metabolismo , Sinapses/patologia , Transmissão Sináptica
15.
J Med Genet ; 52(12): 797-803, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26424145

RESUMO

BACKGROUND: The genetic aetiology of neurodevelopmental defects is extremely diverse, and the lack of distinctive phenotypic features means that genetic criteria are often required for accurate diagnostic classification. We aimed to identify the causative genetic lesions in two families in which eight affected individuals displayed variable learning disability, spasticity and abnormal gait. METHODS: Autosomal recessive inheritance was suggested by consanguinity in one family and by sibling recurrences with normal parents in the second. Autozygosity mapping and exome sequencing, respectively, were used to identify the causative gene. RESULTS: In both families, biallelic loss-of-function mutations in HACE1 were identified. HACE1 is an E3 ubiquitin ligase that regulates the activity of cellular GTPases, including Rac1 and members of the Rab family. In the consanguineous family, a homozygous mutation p.R219* predicted a truncated protein entirely lacking its catalytic domain. In the other family, compound heterozygosity for nonsense mutation p.R748* and a 20-nt insertion interrupting the catalytic homologous to the E6-AP carboxyl terminus (HECT) domain was present; western blot analysis of patient cells revealed an absence of detectable HACE1 protein. CONCLUSION: HACE1 mutations underlie a new autosomal recessive neurodevelopmental disorder. Previous studies have implicated HACE1 as a tumour suppressor gene; however, since cancer predisposition was not observed either in homozygous or heterozygous mutation carriers, this concept may require re-evaluation.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/deficiência , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genes Recessivos , Humanos , Lactente , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Síndrome , Ubiquitina-Proteína Ligases/genética
16.
Nat Cell Biol ; 17(8): 1074-1087, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26167768

RESUMO

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.


Assuntos
Cílios/genética , Transtornos da Motilidade Ciliar/genética , Marcadores Genéticos , Testes Genéticos/métodos , Genômica/métodos , Células Fotorreceptoras , Interferência de RNA , Anormalidades Múltiplas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Doenças Cerebelares/genética , Cerebelo/anormalidades , Cílios/metabolismo , Cílios/patologia , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Bases de Dados Genéticas , Síndrome de Ellis-Van Creveld/genética , Anormalidades do Olho/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Renais Císticas/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reprodutibilidade dos Testes , Retina/anormalidades , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/metabolismo , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
17.
BMC Med Genet ; 16: 8, 2015 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-25928877

RESUMO

BACKGROUND: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. METHODS: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. RESULTS: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. CONCLUSIONS: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations.


Assuntos
Anormalidades Múltiplas/genética , Caseína Quinase I/genética , Fissura Palatina/genética , Exoftalmia/genética , Proteínas da Matriz Extracelular/genética , Microcefalia/genética , Anormalidades da Boca/complicações , Mutação , Osteosclerose/genética , Linhagem , Fenótipo , Anormalidades Dentárias/complicações , Adolescente , Sequência de Bases , Criança , Pré-Escolar , Fissura Palatina/complicações , Exoftalmia/complicações , Feminino , Humanos , Masculino , Microcefalia/complicações , Osteosclerose/complicações , Adulto Jovem
18.
PLoS One ; 9(8): e104281, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25133751

RESUMO

PURPOSE: Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. METHODS: Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. RESULTS: Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). CONCLUSIONS: Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.


Assuntos
Análise Mutacional de DNA/métodos , Distrofias Retinianas/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Linhagem
19.
Nat Genet ; 46(5): 510-515, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24705253

RESUMO

Activating mutations in genes encoding phosphatidylinositol 3-kinase (PI3K)-AKT pathway components cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH, OMIM 603387). Here we report that individuals with MPPH lacking upstream PI3K-AKT pathway mutations carry de novo mutations in CCND2 (encoding cyclin D2) that are clustered around a residue that can be phosphorylated by glycogen synthase kinase 3ß (GSK-3ß). Mutant CCND2 was resistant to proteasomal degradation in vitro compared to wild-type CCND2. The PI3K-AKT pathway modulates GSK-3ß activity, and cells from individuals with PIK3CA, PIK3R2 or AKT3 mutations showed similar CCND2 accumulation. CCND2 was expressed at higher levels in brains of mouse embryos expressing activated AKT3. In utero electroporation of mutant CCND2 into embryonic mouse brains produced more proliferating transfected progenitors and a smaller fraction of progenitors exiting the cell cycle compared to cells electroporated with wild-type CCND2. These observations suggest that cyclin D2 stabilization, caused by CCND2 mutation or PI3K-AKT activation, is a unifying mechanism in PI3K-AKT-related megalencephaly syndromes.


Assuntos
Anormalidades Múltiplas/genética , Ciclina D2/genética , Hidrocefalia/genética , Malformações do Desenvolvimento Cortical/genética , Megalencefalia/genética , Polidactilia/genética , Animais , Sequência de Bases , Western Blotting , Bromodesoxiuridina , Eletroporação , Exoma/genética , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Síndrome
20.
Nat Genet ; 46(4): 326-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24614073

RESUMO

Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.


Assuntos
Colestase Intra-Hepática/genética , Mutação/genética , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-2/genética , Animais , Sequência de Bases , Colestase Intra-Hepática/fisiopatologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Especificidade da Espécie , Junções Íntimas/genética
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