RESUMO
The present study investigates the spectrum and analysis of mitochondrial DNA (mtDNA) variants associated with Leber hereditary optic neuropathy (LHON) in an Argentinean cohort, analyzing 3 LHON-associated mitochondrial genes. In 32% of the cases, molecular confirmation of the diagnosis could be established, due to the identification of disease-causing variants. A total of 54 variants were observed in a cohort of 100 patients tested with direct sequencing analysis. The frequent causative mutations m.11778G>A in MT-ND4, m.3460G>A in MT-ND1, and m.14484T>C in MT-ND6 were identified in 28% of the cases of our cohort. Secondary mutations in this Argentinean LHON cohort were m.11253T>C p.Ile165Thr in MT-ND4, identified in three patients (3/100, 3%) and m.3395A>G p.Tyr30Cys in MT-ND1, in one of the patients studied (1%). This study shows, for the first time, the analysis of mtDNA variants in patients with a probable diagnosis of LHON in Argentina. Standard molecular methods are an effective first approach in order to achieve genetic diagnosis of the disease, leaving NGS tests for those patients with negative results.
Assuntos
DNA Mitocondrial , Atrofia Óptica Hereditária de Leber , Humanos , DNA Mitocondrial/genética , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Argentina , Mitocôndrias/genética , MutaçãoRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.
Assuntos
Heterozigoto , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Inativação do Cromossomo X/genética , Adolescente , Adulto , Feminino , Humanos , Adulto JovemRESUMO
INTRODUCTION: Duchenne/Becker muscular dystrophies (DMD/BMD) are X-linked recessive diseases caused by mutations in the dystrophin gene. METHODS: We used multiplex polymerase chain reaction (PCR) and short tandem repeat (STR) segregation analysis for DMD/BMD-carrier detection and prenatal diagnosis. RESULTS: Twenty-four at-risk pregnancies were evaluated: 17 were excluded from carrying dystrophin gene mutations with 95-100% certainty. Of the remaining cases, 2 were determined to carry a dystrophin gene mutation with 95-100% certainty. Three cases had a 67% probability of carrying the mutation, and 2 others were not informative. The certainty of the test increased to ~100% in some cases due to the identification of several genetic events: 4 recombinations; 4 de novo mutations; and 8 deletions encompassing some of the STRs evaluated. DISCUSSION: Overall, 19 of 24 (79%) molecular prenatal diagnoses were informative, indicating that multiplex PCR/STR segregation analysis is a reliable method for carrier detection and prenatal diagnosis when other more sophisticated techniques are unavailable.
Assuntos
Repetições de Microssatélites/genética , Diagnóstico Pré-Natal/métodos , Argentina , Distrofina/genética , Feminino , Haplótipos/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Mutação/genética , Linhagem , Reação em Cadeia da Polimerase/métodos , GravidezRESUMO
Neurofibromatosis 2 is a familial syndrome characterized by the development of schwannomas, meningiomas and ependymomas. Most of them are benign however, their location in the nervous system has harmful effects on important cranial and spinal structures. These tumors are developed as the outcome of NF2 gene (22q12) inactivation. The NF2 protein, merlin or schwannomin belongs to the Ezrin, Radixin, Moesin (ERM) family involved in the cytoskeletal network and has a tumor suppressor function. Inactivating mutations occur as "de novo" (more frequently) or as inherited, and most of them are frameshift or nonsense. Our aim is to study NF2 gene alterations in Argentine patients and relate them to clinical features. 10 families and 29 single patients were analyzed for: 1) at-risk haplotype by STR-segregation analysis and 2) NF2 gene mutations by SSCP/heteroduplex/sequencing. The at-risk haplotype was uncovered in 8 families and mutations were identified in 5 patients. The molecular data are in full agreement with the clinical features supporting previous reports. The obtained results were important for the detection of mutation-carrying relatives and exclusion of other individuals from risk.
Assuntos
Neurofibromatose 2/genética , Neurofibromina 2/genética , Adolescente , Adulto , Idoso , Argentina , Criança , Ependimoma/genética , Ependimoma/fisiopatologia , Feminino , Haplótipos , Humanos , Masculino , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/fisiopatologia , Meningioma/genética , Meningioma/fisiopatologia , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Mutação , Neurofibromatose 2/fisiopatologia , Linhagem , Adulto JovemRESUMO
We report a Becker muscular dystrophy (BMD) family with one 5-year-old affected patient and a 69-year-old asymptomatic grandfather. Dystrophin gene multiplex polymerase chain reaction and multiplex ligation-dependant probe amplification analysis showed that both males carried an in-frame deletion of exons 45-55. Segregation analysis revealed two additional asymptomatic boys in this family. Our finding supports previous predictions that exons 45-55 are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype.
Assuntos
Distrofina/genética , Éxons/genética , Saúde da Família , Distrofia Muscular de Duchenne/genética , Deleção de Sequência/genética , Idoso , Pré-Escolar , Feminino , Dosagem de Genes , Humanos , Masculino , Análise de SequênciaRESUMO
BACKGROUND: Retinoblastoma (RB) is a malignant tumor originating in the retinal cell precursors and can be presented as a unilateral or bilateral form in childhood (one or both eyes affected). Development of this tumor is caused by mutations in the RB1 gene on chromosome 13q14; the first mutation may occur in the germ line (hereditary RB) or in somatic cells (non-hereditary RB). The hereditary form of RB is transmitted with a high penetrance to offspring (90%). Because early diagnosis is necessary for implementing effective treatment and preserving vision, it is important to identify the mutations in the affected family. AIM: The aim of this study was to identify large and small RB1 germ-line mutations and to correlate them with the RB phenotype. METHODS: Constitutional RB1 gene gross deletions were studied in 40 patients with bilateral or unilateral familial RB by a segregation assay of four intragenic polymorphisms located in introns 1, 4, 17, and 20 of the RB1 gene, along with fluorescence in situ hibridization (FISH) analysis. Small mutations were ascertained in a subgroup of ten patients by heteroduplex/sequence analysis of RB1-exons. RESULTS: In the course of our study, we have found three large deletions, which probably represent whole gene deletions, and two small deletions of 1bp in length. One large deletion was found in a family with several members affected. This represents a rare case of familial RB, which is usually caused by small mutations. Phenotype analysis of the family revealed a low penetrance inheritance, with an 'affected eyes : number of mutation-carriers' ratio of approximately 1.0, whereas this ratio in families with small loss-of-function mutations is 1.5-2.0. CONCLUSIONS: Our results emphasize the usefulness of a combined methodology that includes segregation of polymorphisms, FISH, and heteroduplex/sequence analyses for detection of gross and small DNA rearrangements in familial and sporadic RB. Identification of mutations in sporadic cases is important for risk-assessment in patients' relatives. The degree of penetrance in the inheritance of RB not only depends on the occurrence of the second mutation in the RB1 gene but also on the extent of inactivation of the first mutation.
Assuntos
Neoplasias Oculares/genética , Deleção de Genes , Mutação em Linhagem Germinativa , Proteína do Retinoblastoma/genética , Retinoblastoma/genética , Argentina , Cromossomos Humanos Par 13 , Éxons , Lateralidade Funcional , Humanos , Repetições MinissatélitesRESUMO
BACKGROUND: Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked diseases caused by mutations in the dystrophin gene at Xp21.2; they include gross deletions (60%), duplications (10%), and small mutations (30%). Since there is no cure or effective treatment for progressive muscular dystrophy, prevention of the disease is important and strongly depends on carrier-status information. Two-thirds of DMD/BMD cases are familial; thus, female relatives are candidates for carrier-risk assessment. AIM: Segregation analysis of polymorphic short tandem (CA)n repeats [STR-(CA)n] was used to establish and compare the haplotypes of female relatives of patients with DMD/BMD with those of the patient in order to identify the mutant dystrophin gene and thus determine each female relative's carrier status. METHODS: 248 individuals from 52 families were studied through segregation of up to 11 STR-(CA)n loci. The assay was performed on leukocyte DNA by PCR amplification, polyacrylamide-gel electrophoresis and autoradiography. Haplotypes were established by determination of alleles on the autoradiography. RESULTS: 38 of 51 (75%) female relatives from familial cases were diagnosed as carriers or non-carriers with a 95-100% likelihood, and 18 out of 56 (32%) female relatives from sporadic cases could be excluded from the risk of being a DMD carrier with the same probability. In addition, STR studies detected gross deletions in 13 of the 52 (25%) families in both male and female individuals, four of which were de novo deletions. STR assays were also informative in families without an available DNA sample of an affected male and in two of seven symptomatic females. Determination of carrier status was particularly significant for prediction of DMD risk in prenatal analysis of five male chorionic villi. Other genetic events revealed by STR analysis were: (i) 11 recombinations identified in 6.6% of meiosis in the DMD families; (ii) germinal mosaicism detected in two female carriers; and (iii) changes in STR-(CA)n length during transmission from father to daughters, including three retractions and one elongation at an estimated rate of 0.004. CONCLUSION: The STR assay is an excellent molecular tool for carrier-status identification and the detection of deletions and other genetic changes in families affected by DMD/BMD. Thus, it is useful in genetic counseling for the prevention of this disease.
Assuntos
Portador Sadio/diagnóstico , Distrofia Muscular de Duchenne/genética , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Natal/métodos , Sequência de Bases , Primers do DNA , Repetições de Dinucleotídeos , Feminino , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/embriologia , Linhagem , Polimorfismo Genético , Gravidez , Sequências de Repetição em TandemRESUMO
BACKGROUND: Approximately one-third of new cases of Duchenne muscular dystrophy (DMD) can be attributed to sporadically arising new mutations, however in the majority of cases the DMD mutation has been inherited from the mother. These female carriers can have either a constitutive or mosaic mutation. AIM: The aim of this study was to determine the segregation of the at-risk haplotype and to find a deletion in the dystrophin gene of patients. METHOD: We analyzed individuals from two families with a history of DMD in order to predict the carrier status of related females. In one of these cases the mother had two affected sons, while in the other one son and two grandchildren were affected; therefore we predict that the mother would be an obligatory carrier. RESULTS: Haplotype analysis of the DMD loci revealed that in the two families both the healthy and affected brothers had inherited the same X maternal chromosome. However, the affected brother carried a deletion, which was absent in the unaffected sibling. CONCLUSION: These findings suggested that the mothers in the two families were germline mosaics for the DMD gene. The results of this study demonstrate the usefulness of the methodology that combine the haplotype analysis with the identification of the mutation in order to detect hidden germline mosaicisms and, thus, improve genetic counseling.
Assuntos
Repetições de Dinucleotídeos/genética , Distrofina/genética , Mosaicismo , Distrofia Muscular de Duchenne/diagnóstico , Éxons/genética , Feminino , Deleção de Genes , Mutação em Linhagem Germinativa/genética , Haplótipos/genética , Humanos , Masculino , Linhagem , Polimorfismo GenéticoRESUMO
Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44 %) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8 %) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.
Assuntos
Síndrome de Angelman/diagnóstico , Síndrome de Prader-Willi/diagnóstico , Adolescente , Síndrome de Angelman/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 15/genética , Metilação de DNA , Feminino , Deleção de Genes , Humanos , Lactente , Recém-Nascido , Masculino , Polimorfismo Genético , Síndrome de Prader-Willi/genética , Dissomia Uniparental/genéticaRESUMO
Constitutional RB1 gene mutations were studied in a series of 21 families with unilateral and bilateral retinoblastoma patients. Peripheral blood lymphocytes were analyzed by "exon by exon" PCR-heteroduplex and sequencing. Mutations were identified in 6 (29%) of the patients. One mutation corresponded to an intronic polymorphism in g.174351T > A. The other five mutations resulted C to T exonic transitions, four were CGA sequences (g.65386, g.150037 in two patients, and g.162237), creating stop codons and presumably truncated proteins. The fifth one was new and resulted in alanine to valine substitution (g.73774). Two patients had the same the germline truncated mutation (g.150037C > T), one with a familial bilateral early onset retinoblastoma and one with a sporadic unilateral late onset retinoblastoma. The later type has not been previously described. This finding is discussed in the genotype/phenotype correlation context. Additionally, a single nucleotide change was found in six studied samples, where a C to T homozygous transversion was identified in intron 26 (IVS26 + 28). It is worthy the non concordance of the nucleotide with the published sequence. This analysis proved to be a useful method for the detection of mutations in the RB1 gene, and contributed to the adequate genetic counseling to patients and relatives.
Assuntos
Mutação em Linhagem Germinativa , Penetrância , Retinoblastoma/genética , Idade de Início , Substituição de Aminoácidos , Argentina , Pareamento Incorreto de Bases , Códon sem Sentido , Códon de Terminação , Análise Mutacional de DNA , Feminino , Análise Heteroduplex , Humanos , Íntrons , Masculino , Linhagem , Polimorfismo Genético , Retinoblastoma/química , Retinoblastoma/fisiopatologia , Valina/metabolismoRESUMO
Analyses of deletions in the dystrophin gene and of cognitive status were performed on patients with Duchenne (DMD) or Becker (BMD) muscular dystrophy in order to find a correlation between both features. Molecular study by multiplex and simplex PCR of dystrophin exons led to the identification of 51 deletions in 126 unrelated patients. Most of them were frameshift, in full agreement with severe clinical symptoms, three patients with a BMD-like phenotype had in-frame mutations. Deletions were localized with reference to the different dystrophin isoform sequences and were clustered in two main areas, 5' and central+ 3' end of the gene. Cognitive abilities were tested in 47 out of 51 patients with identified mutations, 23 of them being mentally impaired. Comparison of molecular and neuropsychological features showed that deletions localized in central and 3' parts of the gene (18 out of 23) are preferentially associated with mental impairment. Fourteen of them were found in the regulatory and coding sequences for the three CNS specific carboxy terminal isoforms. Therefore, though mutations with variable locations may lead to cognitive impairment, our results show that deletions in the distal portion of the gene are basically related to mental retardation.
Assuntos
Transtornos Cognitivos/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutação/genética , Transtornos Cognitivos/complicações , Transtornos Cognitivos/fisiopatologia , Éxons/genética , Mutação da Fase de Leitura/genética , Deleção de Genes , Humanos , Deficiência Intelectual/genética , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/metabolismo , Testes Neuropsicológicos , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease. It is inherited as an X-linked recessive trait in which males show clinical manifestations. In some rare cases, the disease can also be manifested in females. The aim of the present study was to determine the molecular alteration in two cases of nonrelated DMD symptomatic carriers with no previous history of DMD. Multiplex PCR is commonly used to search for deletion in the DMD gene of affected males. This method could not be used in females because the normal X chromosome masks the deletion of the mutated one. Therefore, we used a set of seven highly polymorphic dinucleotide (CA)(n) repeat markers that lie within the human dystrophin gene. The deletions were evidenced by hemizygosity of the loci under study. We localized a deletion in the locus 7A (intron 7) on the maternal X chromosome in one case, and a deletion in the region of introns 49 and 50 on the paternal X chromosome in the other. The use of microsatellite genotyping within the DMD gene enables the detection of the mutant allele in female carriers. It is also a useful method to provide DMD families with more accurate genetic counseling.
Assuntos
Análise Mutacional de DNA/métodos , Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/genética , Polimorfismo Genético , Adolescente , Adulto , Sequência de Bases , DNA/química , Primers do DNA/química , Repetições de Dinucleotídeos , Feminino , Triagem de Portadores Genéticos , Humanos , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/diagnóstico , Linhagem , Reação em Cadeia da Polimerase , Sequências de Repetição em TandemRESUMO
The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.