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1.
Hum Mutat ; 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502745

RESUMO

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high-throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3-year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy-number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high-throughput sequencing works as an efficient and cost-effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.

2.
Genet Med ; 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31363182

RESUMO

PURPOSE: Kabuki syndrome (KS) (OMIM 147920 and 300867) is a rare genetic disorder characterized by specific facial features, intellectual disability, and various malformations. Immunopathological manifestations seem prevalent and increase the morbimortality. To assess the frequency and severity of the manifestations, we measured the prevalence of immunopathological manifestations as well as genotype-phenotype correlations in KS individuals from a registry. METHODS: Data were for 177 KS individuals with KDM6A or KMT2D pathogenic variants. Questionnaires to clinicians were used to assess the presence of immunodeficiency and autoimmune diseases both on a clinical and biological basis. RESULTS: Overall, 44.1% (78/177) and 58.2% (46/79) of KS individuals exhibited infection susceptibility and hypogammaglobulinemia, respectively; 13.6% (24/177) had autoimmune disease (AID; 25.6% [11/43] in adults), 5.6% (10/177) with ≥2 AID manifestations. The most frequent AID manifestations were immune thrombocytopenic purpura (7.3% [13/177]) and autoimmune hemolytic anemia (4.0% [7/177]). Among nonhematological manifestations, vitiligo was frequent. Immune thrombocytopenic purpura was frequent with missense versus other types of variants (p = 0.027). CONCLUSION: The high prevalence of immunopathological manifestations in KS demonstrates the importance of systematic screening and efficient preventive management of these treatable and sometimes life-threatening conditions.

3.
Clin Genet ; 95(3): 384-397, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30614526

RESUMO

Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associated with retinal dystrophy, obesity, postaxial polydactyly, learning disabilities, hypogonadism and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly are the hallmark signs of BBS to consider in absence of familial history. However, these findings are not specific to BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (22 known genes). We report here the largest cohort of BBS fetuses to better characterize the antenatal presentation. Prenatal ultrasound (US) and/or autopsy data from 74 fetuses with putative BBS diagnosis were collected out of which molecular diagnosis was established in 51 cases, mainly in BBS genes (45 cases) following the classical gene distribution, but also in other ciliopathy genes (6 cases). Based on this, an updated diagnostic decision tree is proposed. No genotype/phenotype correlation could be established but postaxial polydactyly (82%) and renal cysts (78%) were the most prevalent symptoms. However, autopsy revealed polydactyly that was missed by prenatal US in 55% of the cases. Polydactyly must be carefully looked for in pregnancies with apparently isolated renal anomalies in fetuses.

4.
Fetal Diagn Ther ; : 1-10, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30121677

RESUMO

BACKGROUND: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). METHODS: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. RESULTS: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. CONCLUSIONS: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

5.
Am J Med Genet A ; 176(8): 1760-1763, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30055030

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) has been shown to be related to genetic and epigenetic derepression of DUX4 (mapping to chromosome 4), a gene located within a repeat array of D4Z4 sequences of polymorphic length. FSHD type 1 (FSHD1) is associated with pathogenic D4Z4 repeat array contraction, while FSHD type 2 (FSHD2) is associated with SMCHD1 variants (a chromatin modifier gene that maps to the short arm of chromosome 18). Both FSHD types require permissive polyadenylation signal (4qA) downstream of the D4Z4 array.

6.
Exp Dermatol ; 2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29952037

RESUMO

In ATP6V0A2-related cutis laxa, the skin phenotype varies from a wrinkly skin to prominent cutis laxa and typically associates with skeletal and neurological manifestations. The phenotype remains incompletely characterized, especially in adult patients. Glycosylation defects and reduced acidification of secretory vesicles contribute to the pathogenesis, but the consequences at the clinical level remain to be determined. Moreover, the morphology of the elastic fibres has not been studied in ATP6V0A2-related cutis laxa, nor its relation with potential clinical risks. We report on the extreme variability in ATP6V0A2-related cutis laxa in 10 novel patients, expand the phenotype with emphysema and von Willebrand disease and hypothesize on the pathogenesis that might link both with deficiency of glycosylation and with elastic fibre anomalies. Our data will affect clinical management of patients with ATP6V0A2-related cutis laxa.

7.
Am J Hum Genet ; 101(5): 803-814, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100091

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l-/- embryos and a slight decrease in ureteric bud branching in Greb1l+/- embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.


Assuntos
Anormalidades Congênitas/genética , Nefropatias/congênito , Rim/anormalidades , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas/genética , Animais , Criança , Exoma/genética , Feminino , Feto/anormalidades , Heterozigoto , Humanos , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Sistema Urinário/anormalidades , Anormalidades Urogenitais/genética
8.
J Med Genet ; 54(6): 432-440, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28258187

RESUMO

BACKGROUND: Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded by COL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations in GPR56 give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations in COL3A1 are associated with a similar phenotype. METHODS: Exome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations in COL3A1 were reviewed. Functional assays were performed on dermal fibroblasts. RESULTS: Exome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 of COL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelic COL3A1 mutations showed a brain phenotype similar to that associated with mutations in GPR56. CONCLUSION: Homozygous or compound heterozygous mutations in COL3A1 are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelic COL3A1 mutations emphasises the critical role of the type III collagen-GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.


Assuntos
Colágeno Tipo III/genética , Cistos/genética , Malformações do Desenvolvimento Cortical/genética , Receptores Acoplados a Proteínas-G/genética , Substância Branca/patologia , Adulto , Alelos , Células Cultivadas , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Criança , Pré-Escolar , Cistos/patologia , Exoma/genética , Éxons/genética , Feminino , Fibroblastos/patologia , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Masculino , Malformações do Desenvolvimento Cortical/patologia , Mutação/genética , Fenótipo , Adulto Jovem
9.
Genet Med ; 19(9): 989-997, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28151489

RESUMO

PURPOSE: Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS. METHODS: We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested. RESULTS: We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10-5). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10-25), regardless of the phenotype. CONCLUSION: Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions.Genet Med advance online publication 02 February 2017.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Estudos de Associação Genética , Testes Genéticos , Transtornos do Crescimento/diagnóstico , Transtornos do Crescimento/genética , Mutação , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Gerenciamento Clínico , Feminino , Predisposição Genética para Doença , Testes Genéticos/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Mosaicismo , Fenótipo , Diagnóstico Pré-Natal , Análise de Sequência de DNA , Adulto Jovem
10.
Am J Med Genet A ; 170(11): 2847-2859, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27605097

RESUMO

KBG syndrome, due to ANKRD11 alteration is characterized by developmental delay, short stature, dysmorphic facial features, and skeletal anomalies. We report a clinical and molecular study of 39 patients affected by KBG syndrome. Among them, 19 were diagnosed after the detection of a 16q24.3 deletion encompassing the ANKRD11 gene by array CGH. In the 20 remaining patients, the clinical suspicion was confirmed by the identification of an ANKRD11 mutation by direct sequencing. We present arguments to modulate the previously reported diagnostic criteria. Macrodontia should no longer be considered a mandatory feature. KBG syndrome is compatible with autonomous life in adulthood. Autism is less frequent than previously reported. We also describe new clinical findings with a potential impact on the follow-up of patients, such as precocious puberty and a case of malignancy. Most deletions remove the 5'end or the entire coding region but never extend toward 16q telomere suggesting that distal 16q deletion could be lethal. Although ANKRD11 appears to be a major gene associated with intellectual disability, KBG syndrome remains under-diagnosed. NGS-based approaches for sequencing will improve the detection of point mutations in this gene. Broad knowledge of the clinical phenotype is essential for a correct interpretation of the molecular results. © 2016 Wiley Periodicals, Inc.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/diagnóstico , Doenças do Desenvolvimento Ósseo/genética , Estudos de Associação Genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Proteínas Repressoras/genética , Anormalidades Dentárias/diagnóstico , Anormalidades Dentárias/genética , Adolescente , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 16 , Hibridização Genômica Comparativa , Facies , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Adulto Jovem
12.
J Bone Miner Res ; 30(8): 1369-76, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25656438

RESUMO

Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutation in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1), leading to transforming growth factor-beta (TGF-ß) signaling dysregulation. Although decreased axial and peripheral bone mineral density (BMD) has been reported in adults with MFS, data about the evolution of bone mass during childhood and adolescence are limited. The aim of the present study was to evaluate bone and muscle characteristics in children, adolescents, and young adults with MFS. The study population included 48 children and young adults (22 girls) with MFS with a median age of 11.9 years (range 5.3 to 25.2 years). The axial skeleton was analyzed at the lumbar spine using dual-energy X-ray absorptiometry (DXA), whereas the appendicular skeleton (hand) was evaluated using the BoneXpert system (with the calculation of the Bone Health Index). Muscle mass was measured by DXA. Compared with healthy age-matched controls, bone mass at the axial and appendicular levels and muscle mass were decreased in children with MFS and worsened from childhood to adulthood. Vitamin D deficiency (<50 nmol/L) was found in about a quarter of patients. Serum vitamin D levels were negatively correlated with age and positively correlated with lumbar spine areal and volumetric BMD. Lean body mass (LBM) Z-scores were positively associated with total body bone mineral content (TB-BMC) Z-scores, and LBM was an independent predictor of TB-BMC values, suggesting that muscle hypoplasia could explain at least in part the bone loss in MFS. Patients with a FBN1 premature termination codon mutation had a more severe musculoskeletal phenotype than patients with an inframe mutation, suggesting the involvement of TGF-ß signaling dysregulation in the pathophysiologic mechanisms. In light of these results, we recommend that measurement of bone mineral status should be part of the longitudinal clinical investigation of MFS children.


Assuntos
Envelhecimento , Genótipo , Síndrome de Marfan , Proteínas dos Microfilamentos/genética , Músculo Esquelético , Coluna Vertebral , Absorciometria de Fóton , Adulto , Envelhecimento/genética , Envelhecimento/patologia , Densidade Óssea , Criança , Pré-Escolar , Códon de Terminação , Estudos Transversais , Feminino , Fibrilina-1 , Fibrilinas , Seguimentos , Humanos , Masculino , Síndrome de Marfan/diagnóstico por imagem , Síndrome de Marfan/genética , Síndrome de Marfan/fisiopatologia , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/fisiopatologia , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/fisiopatologia
13.
Orphanet J Rare Dis ; 9: 207, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25497157

RESUMO

PMM2-CDG (formerly known as CDG Ia) a deficiency in phosphomannomutase, is the most frequent congenital disorder of glycosylation. The phenotype encompasses a wide range of neurological and non-neurological manifestations comprising cerebellar atrophy and intellectual deficiency. The phenotype of the disorder is well characterized in children but the long term course of the disease is unknown and the phenotype of late onset forms has not been comprehensively described. We thus retrospectively collected the clinical, biological and radiological data of 29 French PMM2-CDG patients aged 15 years or more with a proven molecular diagnosis (16 females and 13 males). In addition, thirteen of these patients were reexamined at the time of the study to obtain detailed information. 27 of the 29 patients had a typical PMM2-CDG phenotype, with infantile hypotonia, strabismus, developmental delay followed by intellectual deficiency, epilepsy, retinitis pigmentosa and/or visceral manifestations. The main health problems for these patients as teenagers and in adulthood were primary ovarian insufficiency, growth retardation, coagulation anomalies and thrombotic events, skeletal deformities and osteopenia/osteoporosis, retinitis pigmentosa, as well as peripheral neuropathy. Three patients had never walked and three lost their ability to walk. The two remaining patients had a late-onset phenotype unreported to date. All patients (n = 29) had stable cerebellar atrophy. Our findings are in line with those of previous adult PMM2-CDG cohorts and points to the need for a multidisciplinary approach to the follow up of PMM2-CDG patients to prevent late complications. Additionally, our findings add weight to the view that PMM2-CDG may be diagnosed in teenage/adult patients with cerebellar atrophy, even in the absence of intellectual deficiency or non-neurological involvement.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Fenótipo , Fosfotransferases (Fosfomutases)/deficiência , Adolescente , Adulto , Idade de Início , Estudos de Coortes , Defeitos Congênitos da Glicosilação/epidemiologia , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fosfotransferases (Fosfomutases)/genética , Estudos Retrospectivos , Adulto Jovem
14.
Am J Hum Genet ; 94(2): 288-94, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24439109

RESUMO

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.


Assuntos
Anormalidades Congênitas/genética , Genes Recessivos , Cadeias alfa de Integrinas/genética , Nefropatias/congênito , Rim/anormalidades , Anormalidades Urogenitais/genética , Anormalidades Congênitas/patologia , Feminino , Feto/anormalidades , Homozigoto , Humanos , Cadeias alfa de Integrinas/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Masculino , Mutação , Linhagem , Anormalidades Urogenitais/patologia
15.
Hum Mutat ; 34(8): 1080-4, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23649991

RESUMO

Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication-triplication rearrangement involving exons 45-60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) events; specifically, a 690-kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46-kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X-Y chromosome recombination during male meiosis, we proposed an alternative two-step model for the generation of this X-linked DMD DUP-TRP/INV-DUP event.


Assuntos
Distrofina/genética , Duplicação Gênica , Distrofia Muscular de Duchenne/genética , Adolescente , Sequência de Bases , Quebras de DNA , Variações do Número de Cópias de DNA , Distrofina/metabolismo , Éxons , Variação Genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Sequências Repetidas Invertidas , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/metabolismo , Inversão de Sequência
16.
Genet Res Int ; 2013: 784789, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24386570

RESUMO

Background. Venous malformations (VM) result from an error in vascular morphogenesis. The first gene suspected in their development is the TEK gene (tyrosine kinase, endothelial). Mutations of this gene have been identified in several Belgian families with a dominant form of the disease. Therefore, we investigated whether mutations in this TEK gene could explain the MV development in patients of families from Tlemcen region (north-western Algeria). Methods. Genomic DNA was extracted from leucocytes of ten patients. The search for mutations in all the 23 exons and in the 5' and 3' intronic sequences flanking the TEK gene was performed using PCR amplification and direct sequencing of amplified genomic DNA. Additionally, a search for somatic mutations of the gene TEK was performed on a biopsy of the venous malformation from one of the ten eligible patients. Results. The sequencing of the 23 exons of the TEK gene revealed neither germinal mutation in our ten patients nor somatic mutation in the tissue of the biopsy. Conclusion. The absence of mutation in the TEK gene in the population studied suggests that the TEK gene is not necessarily involved in the onset of VM; its association with these malformations may differ from one population to another.

17.
Virchows Arch ; 460(6): 637-49, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22549280

RESUMO

Vascular Ehlers-Danlos syndrome (vEDS) results from a mutation in the gene encoding alpha-1, type III pro-collagen (COL3A1) and confers fragility to skin, ligament and vascular tissue. We tested the value of skin biopsy for diagnosis of vEDS through an ultrastructure scoring procedure. Study design was a multicentric, case-control, blinded trial consisting of two phases: phase 1 was to identify an ultra-structure score providing the best discriminative value for vEDS and phase 2 was to replicate this result in a different population. We enrolled 103 patients, 66 cases defined through the revised nosology for Ehlers-Danlos syndromes and 37 control subjects selected from patients referred for other pathologies. Ultrastructure of extracellular matrix was read by three to five experienced pathologists blinded for diagnosis. We used the receiver operating curves and logistic regression analysis for ranking ultrastructure scores. We created a detailed description of lesions observed in vEDS patients with 27 items (coded 0 or 1). In the phase 1 (17 cases and 20 controls), abnormal fibroblast shape, presence of lysosomes in the fibroblast and abnormal basal lamina were found to be independent discriminative items. Addition of these three items (defining an ultrastructure score) had the best diagnosis value (area under the curve (AUC) = 0.96). In the phase 2 (49 cases, 17 controls), ultrastructure score provided odds ratio of 9.76 (95 % CI 2.91-32.78), and AUC of 0.90. The ultrastructure score of skin biopsy has predictive value for the diagnosis of vEDS. Presence of two or more signs (either abnormal fibroblast, presence of lysosomes in the fibroblast or abnormal basal lamina) is very evocative of vEDS.


Assuntos
Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/diagnóstico , Pele/ultraestrutura , Biópsia , Colágeno Tipo III/ultraestrutura , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , Humanos
18.
Eur J Hum Genet ; 20(10): 1096-100, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22510846

RESUMO

We report on the effectiveness of a custom-designed oligonucleotide-based comparative genomic hybridization microarray (array-CGH) to interrogate copy number across the entire 2.2-Mb genomic region of the DMD gene and its applicability in diagnosis. The high-resolution array-CGH, we developed, successfully detected a series of 42 previously characterized large rearrangements of various size, localization and type (simple or complex deletions, duplications, triplications) and known intronic CNVs/Indels. Moreover, the technique succeeded in identifying a small duplication of only 191 bp in one patient previously negative for DMD mutation. Accurate intronic breakpoints localization by the technique enabled subsequent junction fragments identification by sequencing in 86% of cases (all deletion cases and 62.5% of duplication cases). Sequence examination of the junctions supports a role of microhomology-mediated processes in the occurrence of DMD large rearrangements. In addition, the precise knowledge of the sequence context at the breakpoints and analysis of the resulting consequences on maturation of pre-mRNA contribute to elucidating the cause of discrepancies in phenotype/genotype correlations in some patients. Thereby, the array-CGH proved to be a highly efficient and reliable diagnostic tool, and the new data it provides will have many potential implications in both, clinics and research.


Assuntos
Hibridização Genômica Comparativa , Distrofina/genética , Aberrações Cromossômicas , Pontos de Quebra do Cromossomo , Variações do Número de Cópias de DNA , Feminino , Humanos , Íntrons , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética
19.
Hum Mutat ; 32(4): 467-75, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21305657

RESUMO

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double-deletion events that possibly involve microhomology-mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387-bp PE was originated from an inverted ∼57 kb genomic region of intron 44 flanked by two deleted ∼52 kb and ∼1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166-bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases.


Assuntos
Distrofina/genética , Éxons/genética , Íntrons/genética , Distrofia Muscular de Duchenne/genética , Mutação , Adulto , Pré-Escolar , Rearranjo Gênico , Humanos , Masculino , Processamento de RNA
20.
Am J Med Genet A ; 152A(6): 1550-4, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20503334

RESUMO

This report describes two unrelated boys presenting with short stature, femoral metaphyseal abnormalities, platyspondyly, and retinitis pigmentosa. Patients share similar findings with cases described by Ehara et al. [Ehara et al. (1997); Eur J Pediatr 156:627-630] described as axial spondylometaphyseal dysplasia. The presence of consanguinity in one of our patients further supports an autosomal recessive mode of inheritance of what, we believe, constitutes a separate and distinct entity.


Assuntos
Fêmur/anormalidades , Transtornos do Crescimento/diagnóstico , Osteocondrodisplasias/diagnóstico , Degeneração Retiniana/diagnóstico , Retinite Pigmentosa/diagnóstico , Adolescente , Criança , Transtornos do Crescimento/genética , Humanos , Masculino , Osteocondrodisplasias/diagnóstico por imagem , Osteocondrodisplasias/genética , Radiografia , Degeneração Retiniana/genética , Retinite Pigmentosa/genética , Síndrome
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