RESUMO
BACKGROUND: Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant syndrome predisposing to the early development of various cancers including those of colon, rectum, endometrium, ovarium, small bowel, stomach and urinary tract. HNPCC is caused by germline mutations in the DNA mismatch repair genes, mostly hMSH2 or hMLH1. In this study, we report the analysis for genetic counseling of three first-degree relatives (the mother and two sisters) of a male who died of colorectal adenocarcinoma at the age of 23. The family fulfilled strict Amsterdam-I criteria (AC-I) with the presence of extracolonic tumors in the extended pedigree. We overcame the difficulty of having a proband post-mortem non-tumor tissue sample for MSI testing by studying the alleles carried by his progenitors. METHODS: Tumor MSI testing is described as initial screening in both primary and metastasis tumor tissue blocks, using the reference panel of 5 microsatellite markers standardized by the National Cancer Institute (NCI) for the screening of HNPCC (BAT-25, BAT-26, D2S123, D5S346 and D17S250). Subsequent mutation analysis of the hMLH1 and hMSH2 genes was performed. RESULTS: Three of five microsatellite markers (BAT-25, BAT-26 and D5S346) presented different alleles in the proband's tumor as compared to those inherited from his parents. The tumor was classified as high frequency microsatellite instability (MSI-H). We identified in the HNPCC family a novel germline missense (c.1864C>A) mutation in exon 12 of hMSH2 gene, leading to a proline 622 to threonine (p.Pro622Thr) amino acid substitution. CONCLUSION: This approach allowed us to establish the tumor MSI status using the NCI recommended panel in the absence of proband's non-tumor tissue and before sequencing the obligate carrier. According to the Human Gene Mutation Database (HGMD) and the International Society for Gastrointestinal Hereditary Tumors (InSiGHT) Database this is the first report of this mutation.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Análise Mutacional de DNA/métodos , Mutação em Linhagem Germinativa , Repetições de Microssatélites , Proteína 2 Homóloga a MutS/genética , Adulto , Sequência de Aminoácidos , Análise Mutacional de DNA/normas , Feminino , Testes Genéticos , Instabilidade Genômica , Humanos , Masculino , Dados de Sequência Molecular , National Institutes of Health (U.S.) , Linhagem , Proteínas/genética , Padrões de Referência , Alinhamento de Sequência , Estados UnidosRESUMO
A extração de DNA de leucócitos periféricos é o meio de obtenção de DNA mais amplamente utilizado. Entretanto, a coleta de células a partir de swab oral, geralmente utilizada em medicina forense, é útil para obtenção de amostras de DNA de recém-nascidos, crianças e de pacientes que vivem em locais onde a coleta e o envio da amostra de sangue não é factível. Nosso objetivo foi padronizar a técnica de extração de DNA a partir de swab de células de mucosa oral utilizando NaCl, comparando-a com a extração pelo kit comercial. Para testar a qualidade do DNA, amplificamos os 3 éxons do gene PROP1 de 12 pacientes com hipopituitarismo hipofisário em DNA extraído simultaneamente de células da mucosa oral e de sangue periférico. A amplificação de fragmentos maiores foi testada em DNA de mucosa oral de indivíduos normais utilizando-se primers do éxon 10 do gene do FSHR (1000pb) e do gene CYP21A2 (1200pb). Ambos os métodos resultaram em DNA de boa qualidade, permitindo o estudo molecular. O método por NaCl mostrou-se mais rápido e barato, resultando em maior quantidade de DNA quando comparado ao kit comercial. Nos pacientes com hipopituitarismo, identificamos a mutação delAG301-302 em 6 pacientes, 4 em homozigose (33 por cento) e 2 em heterozigose (16 por cento), e a mutação G51A em heterozigose em uma paciente. Em conclusão, padronizamos a técnica de extração de DNA de células de swab oral com NaCl que, quando comparada à extração com kit comercial, apresentou menor custo e maior rapidez, indicando ser esta uma forma confiável de obtenção de DNA para estudos genéticos.
Assuntos
Humanos , Recém-Nascido , Criança , Análise Mutacional de DNA/normas , Cloreto de Sódio , DNA , Hipopituitarismo/genética , Proteínas de Homeodomínio/genética , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Estudos de Casos e Controles , Amplificação de Genes , Leucócitos/química , Mucosa Bucal/citologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To describe the development and follow-up confirmatory results of the routine cystic fibrosis (CF) newborn screening (NBS) program in Wisconsin. METHODS: CF NBS has been performed on a routine clinical basis in Wisconsin since July 1994. The 2-tiered immunoreactive trypsinogen (IRT)/DNA technique was used on dried blood on filter paper spots. From July 1994 to February 2002, mutation analysis was for the DeltaF508 allele. Beginning in March 2002, multimutation analysis of 25 CF mutations was performed. Infants with a positive result on NBS were seen in certified CF centers for sweat testing by means of quantitative pilocarpine iontophoresis, and families received genetic counseling. RESULTS: From July 1994 to February 2002, there were 120 cases of CF detected by means of NBS (509,794 infants screened), with 53 DeltaF508 homozygotes and 67 compound heterozygotes. There were 8 clinically diagnosed cases of CF (no DeltaF508 allele). The CF incidence was 1:3983 (95%CI, 1:3373-1:4774). From March 2002 to June 2003, multimutation analysis identified 21 cases of classic CF (90,142 infants screened). Sweat tests were successfully performed in infants younger than 1 month. CONCLUSIONS: Early diagnosis of CF through NBS was successfully performed, with an estimated sensitivity rate of 99% using the IRT/25 CFTR multimutation assay.
Assuntos
Fibrose Cística/diagnóstico , Análise Mutacional de DNA/métodos , Fluorimunoensaio/métodos , Triagem Neonatal/métodos , Tripsinogênio/sangue , Fatores Etários , Cloretos/análise , Fibrose Cística/sangue , Fibrose Cística/epidemiologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA/normas , Diagnóstico Precoce , Eletroforese em Gel de Poliacrilamida/métodos , Fluorimunoensaio/normas , Heterozigoto , Homozigoto , Humanos , Incidência , Lactente , Recém-Nascido , Iontoforese/métodos , Agonistas Muscarínicos , Triagem Neonatal/normas , Pilocarpina , Reação em Cadeia da Polimerase/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Sensibilidade e Especificidade , Suor/química , Wisconsin/epidemiologiaRESUMO
OBJECTIVE: To quantitate the proportion of infants identified through cystic fibrosis (CF) newborn screening (NBS) by an immunoreactive trypsinogen (IRT)/DNA screening algorithm who have an unclear diagnosis as defined by the findings of an elevated IRT level and either 1) 2 CF gene (CFTR) mutations detected and sweat chloride level <60 mEq/L; or 2) 0 or 1 CFTR mutations and a "borderline" sweat chloride level >or=30 and <60 mEq/L. STUDY DESIGN: Using the 4-year cohort of CF-affected infants recently described by the Massachusetts CF NBS program, we identified and described the number of infants with the diagnostic characteristics (diagnostic dilemmas) aforementioned. RESULTS: Of infants with positive results on CF NBS who had 1 CFTR mutation detected and a borderline sweat chloride concentration, nearly 20% displayed a second CFTR mutation on further evaluation. Of all infants with positive CF NBS results considered affected with CF, 11% had a diagnosis that fell into 1 of the diagnostic dilemma categories aforementioned. CONCLUSIONS: Four problematic diagnostic categories generated by CF NBS are defined. In the absence of data on the natural history of such infants, careful follow-up is recommended for infants in whom a definitive diagnosis is elusive.
Assuntos
Algoritmos , Fibrose Cística/diagnóstico , Análise Mutacional de DNA/métodos , Imunoensaio/métodos , Triagem Neonatal/métodos , Tripsinogênio/sangue , Cloretos/análise , Protocolos Clínicos , Fibrose Cística/sangue , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA/normas , Árvores de Decisões , Reações Falso-Negativas , Reações Falso-Positivas , Seguimentos , Humanos , Imunoensaio/normas , Recém-Nascido , Massachusetts , Mutação/genética , Triagem Neonatal/normas , Guias de Prática Clínica como Assunto , Suor/químicaRESUMO
OBJECTIVE: To examine immunoreactive trypsinogen (IRT)-based screening for cystic fibrosis (CF) for recall rate, genotype distribution, and "borderline" sweat test results. STUDY DESIGN: CF newborn screening in Colorado began in 1982, and >1,153,000 infants were screened through 2002 with an IRT-based screen (IRT/IRT). RESULTS: We have identified 313 infants with CF, giving an overall incidence of 1 in 3684 and a Hispanic incidence of 1 in 6495. Fifty-five infants with meconium ileus (17.6%) were excluded from analysis. Fourteen infants with false-negative results were identified (5.4%). The average recall rate was 0.6%, with a positive predictive value of 4.7%. Ninety-three percent of the infants had at least 1 DeltaF508 mutation, and 98% of the infants had at least 1 mutation from the American College of Medical Genetics recommended panel. Six infants had hypertrypsinogenemia and borderline results on sweat tests (30-60 mmol/L). Increased variability in sweat chloride levels were seen in these infants compared with infants with homozygous DeltaF508. Three children with initial borderline results on sweat tests had CF diagnosed. CONCLUSIONS: The recall and false-negative rates of our IRT/IRT CF screening program are reported. Additionally, genotypes of the patients identified mirror the CF population genotypes, reflecting similar disease severity in the screened population. Finally, infants with persistent hypertrypsinogenemia and borderline sweat test results need long-term follow-up.
Assuntos
Fibrose Cística/diagnóstico , Análise Mutacional de DNA/métodos , Fluorimunoensaio/métodos , Triagem Neonatal/métodos , Radioimunoensaio/métodos , Tripsinogênio/sangue , Cloretos/análise , Colorado/epidemiologia , Fibrose Cística/sangue , Fibrose Cística/epidemiologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA/normas , Reações Falso-Negativas , Fluorimunoensaio/normas , Genótipo , Humanos , Incidência , Recém-Nascido , Testes Obrigatórios , Mutação/genética , Triagem Neonatal/normas , Valor Preditivo dos Testes , Radioimunoensaio/normas , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Suor/químicaRESUMO
DNA extraction of peripheral leukocytes is the most broadly used technique to obtain DNA. However, cell collection from an oral swab, frequently used in forensics, is useful to obtain DNA samples, mainly in newborns, children and patients who live far from the collection sites, where blood sample collection and sending is not feasible. Our objective was to standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit. To test DNA quality, we amplified the 3 exons of PROP1 gene of 12 patients with hypopituitarism in DNA obtained from oral cells and peripheral blood cells. Amplification of larger fragments was tested in oral DNA of normal subjects using primers of exon 10 of FSHR gene (1000 bp) and of CYP21A2 gene (1200 bp). Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared to the commercial kit. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In conclusion, we standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that DNA from oral swabs are a reliable source for genetic studies.
Assuntos
Análise Mutacional de DNA/normas , DNA/isolamento & purificação , Proteínas de Homeodomínio/genética , Hipopituitarismo/genética , Cloreto de Sódio , Estudos de Casos e Controles , Criança , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Amplificação de Genes , Humanos , Recém-Nascido , Leucócitos/química , Mucosa Bucal/citologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), one of the most common single gene disorders. It is thought that FH affects approximately 1 of 500 individuals in most populations. Single-strand conformation polymorphism (SSCP) analysis is widely used to detect mutations in the LDLR gene. However, several factors such as temperature, pH, running time, gel composition and size of the DNA fragments can influence its sensitivity. We have optimized the electrophoretic conditions to screen mutations in the promoter region and exons 1-18 of the LDLR gene by varying temperature (5 degrees C, 8 degrees C, 12 degrees C and 15 degrees C), voltage (300 to 600 V), and running time (1 to 4 hours) in the semi-automated GenePhor system (Amersham Biosciences). The efficiency of the method was evaluated by using 30 positive controls (DNA samples with mutations and polymorphisms in the LDLR gene, previously characterized) and DNA samples from 90 Brazilian patients with FH. Our results show that the use of two temperatures (5 degrees C and 15 degrees C) in combination with other optimized conditions resulted in high mutation detection rate (97%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for the diagnosis of genetic disorders, cancer, and for pharmacogenetic studies.