RESUMO
Decompression sickness (DCS) is a systemic syndrome that can occur after an environmental pressure reduction. Previously, we showed that the plasmatic tetrameric form of transthyretin (TTR) nearly disappeared in rats suffering DCS but not in asymptomatic ones. In this pilot study, we assessed whether the resistance to DCS could be associated with polymorphism of the gene of TTR. For this study, Sanger sequencing was performed on purified PCR products from the liver of 14-week-old male and female standard and DCS-resistant rats (n = 5 per group). Hepatic TTR mRNA expression was assessed by RT-qPCR in 18-19 week-old male and female standard and resistant rats (n = 6 per group). There is a synonymous single nucleotide polymorphism (SNP) on the third base of codon 46 (c.138 C > T). The thymine allele was present in 90% and 100% of males and females standard, respectively. However, this allele is present in only 30% of DCS-resistant males and females (p = 0.0002301). In the liver, there is a significant effect of the resistance to DCS (p = 0.043) and sex (p = 0.047) on TTR expression. Levels of TTR mRNA were lower in DCS-resistant animals. To conclude, DCS resistance might be associated with a SNP and a lower expression of TTR.
Assuntos
Doença da Descompressão , Fígado , Polimorfismo de Nucleotídeo Único , Pré-Albumina , Animais , Masculino , Pré-Albumina/genética , Pré-Albumina/metabolismo , Feminino , Projetos Piloto , Ratos , Doença da Descompressão/genética , Doença da Descompressão/metabolismo , Fígado/metabolismo , Resistência à Doença/genética , Ratos Wistar , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
OBJECTIVE: As exercise intolerance and exercise-induced myalgia are commonly encountered in metabolic myopathies, functional screening tests are commonly used during the diagnostic work-up. Our objective was to evaluate the accuracy of isometric handgrip test (IHT) and progressive cycle ergometer test (PCET) to identify McArdle disease and myoadenylate deaminase (MAD) deficiency and to propose diagnostic algorithms using exercise-induced lactate and ammonia variations. METHODS: A prospective sample of 46 patients underwent an IHT and a PCET as part of their exercise-induced myalgia and intolerance evaluation. The two diagnostics tests were compared against the results of muscle biopsy and/or the presence of mutations in PYGM. A total of 6 patients had McArdle disease, 5 a complete MAD deficiency (MAD absent), 12 a partial MAD deficiency, and 23 patients had normal muscle biopsy and acylcarnitine profile (disease control). RESULTS: The two functional tests could diagnose all McArdle patients with statistical significance, combining a low lactate variation (IHT: <1 mmol/L, AUC = 0.963, P < .0001; PCET: <1 mmol/L, AUC = 0.990, P < .0001) and a large ammonia variation (IHT: >100 µmol/L, AUC = 0.944, P = .0005; PCET: >20 µmol/L, AUC = 1). PCET was superior to IHT for MAD absent diagnosis, combining very low ammonia variation (<10 µmol/L, AUC = 0.910, P < .0001) and moderate lactate variation (>1 mmol/L). CONCLUSIONS: PCET-based decision tree was more accurate than IHT, with respective generalized squared correlations of 0.796 vs 0.668. IHT and PCET are both interesting diagnostic tools to identify McArdle disease, whereas cycle ergometer exercise is more efficient to diagnose complete MAD deficiency.
Assuntos
AMP Desaminase/deficiência , Algoritmos , Teste de Esforço/métodos , Doença de Depósito de Glicogênio Tipo V/diagnóstico , Força da Mão/fisiologia , AMP Desaminase/genética , Adolescente , Adulto , Exercício Físico/fisiologia , Feminino , Doença de Depósito de Glicogênio Tipo V/genética , Doença de Depósito de Glicogênio Tipo V/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Estudos Prospectivos , Adulto JovemRESUMO
In October 2014, an outbreak of botulism type D/C occurred on two cattle farms in close proximity. A poultry farm located nearby with no history of botulism had transferred poultry manure to both bovine farms before the beginning of the outbreak. Given this context, epidemiological investigation was conducted to determine if the poultry farm was a reservoir of C. botulinum type D/C and to identify the source of contamination on the cattle farms. Environmental samples were collected at three houses on the poultry farm (boot swabs from the surroundings, swabs from the ventilation system, boot swabs from the poultry litter and darkling beetles samples), and on the two cattle farms (silage samples, boot swabs from the cattle stalls, boot swabs from the cattle pasture and poultry manure samples). These samples were analyzed using real-time PCR after an enrichment step to detect C. botulinum type D/C. On the poultry farm, three boot swabs from the surroundings, two swabs from the ventilation system, one boot swab from the litter and one sample of darkling beetles were detected positive. On one cattle farm, C. botulinum type D/C was identified in a sample of silage made from grass grown on a field on which the poultry manure had previously been stored and in a boot swab from a pasture. On the other cattle farm, C. botulinum type D/C was detected in a sample of poultry manure stored on the cattle farm and in a boot swab from a pasture. This investigation shows that the healthy poultry farm might have been the reservoir of C. botulinum type D/C and that cross-contamination between poultry and cattle likely occurred, resulting in the botulism outbreak on the two cattle farms.
Assuntos
Botulismo/veterinária , Doenças dos Bovinos/etiologia , Galinhas , Surtos de Doenças/veterinária , Doenças das Aves Domésticas/microbiologia , Animais , Botulismo/transmissão , Bovinos , Doenças dos Bovinos/patologia , Clostridium botulinum , Microbiologia Ambiental , Fazendas , Feminino , Masculino , EstercoRESUMO
In 2014, a botulism outbreak in a flock of laying hens was investigated in France. In the flock of 5020 hens, clinical signs of botulism occurred at 46 weeks of age. A type C/D botulism outbreak was confirmed using the mouse lethality assay for detection of botulinum toxin in serum and a real-time PCR test to detect Clostridium botulinum in intestinal contents. The disease lasted one week with a mortality rate of 2.6% without recurrence. Botulism in laying hens has rarely been reported. Five monthly visits were made to the farm between December 2014 and May 2015 for a longitudinal study of the persistence of C. botulinum in the poultry house after the outbreak, and to assess egg contamination by C. botulinum. Several samples were collected on each visit: in the house (from the ventilation circuit, the egg circuit, water and feed, droppings) and the surrounding area. Thirty clean and 30 dirty eggs were also swabbed at each visit. In addition, 12 dirty and 12 clean eggs were collected to analyse eggshell and egg content. The samples were analysed using real-time PCR to detect type C/D C. botulinum. The bacterium was still detected in the house more than 5 months after the outbreak, mostly on the walls and in the egg circuit. Regarding egg contamination, the bacteria were detected only on the shell but not in the content of the eggs. Control measures should therefore be implemented throughout the egg production period to avoid dissemination of the bacteria, particularly during egg collection.
Assuntos
Toxinas Botulínicas/sangue , Botulismo/veterinária , Galinhas/microbiologia , Clostridium botulinum/isolamento & purificação , Surtos de Doenças/veterinária , Doenças das Aves Domésticas/microbiologia , Animais , Botulismo/epidemiologia , Botulismo/microbiologia , Clostridium botulinum/genética , Ovos/microbiologia , Feminino , França/epidemiologia , Estudos Longitudinais , Camundongos , Doenças das Aves Domésticas/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
BACKGROUND: Patients suffering from haemoglobinopathies may be treated by red blood cell (RBC) transfusion on a regular basis and then exposed to multiple antigens with a recurrent, potential risk of alloimmunization routinely prevented by extended RBC antigen cross-matching. While time-consuming and labour-intensive serological analyses are the gold standard for RBC typing, genotyping by current high-throughput molecular tools, including next-generation sequencing (NGS), appears to offer a potent alternative. STUDY DESIGN AND METHODS: The potential of extended blood group genotyping (EBGG) by NGS of 17 genes involved in 14 blood group systems was evaluated in a cohort of 48 patients with sickle-cell disease. Sample preparation and sequencing were simplified and automated for future routine implementation. RESULTS: Sequencing data were obtained for all DNA samples with two different sequencing machines. Prediction of phenotypes could be made in 12 blood group systems and partially in two other blood group systems (Rh and MNS). Importantly, predicted phenotypes in the MNS (S/s), Duffy, Kidd and Kell systems matched well with serological data (98·9%), when available. Unreferenced alleles in the ACHE and ART4 genes, respectively, involved in the Yt and Dombrock blood groups, were identified, then contributing to extend the current knowledge of blood group molecular genetics. CONCLUSIONS: Overall, we consider that our strategy for NGS-based EBGG, assisted by a simple method for genotyping exons 1 and 2 of the pairs of homologous genes (i.e. RHD/RHCE and GYPA/GYPB), as well as the future support of potent bioinformatics tools, may be implemented for routine diagnosis in specific populations.
Assuntos
Anemia Falciforme/genética , Anemia Falciforme/terapia , Sequência de Bases , Tipagem e Reações Cruzadas Sanguíneas , Segurança do Sangue , Análise Mutacional de DNA , Transfusão de Eritrócitos , Frequência do Gene , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sistema do Grupo Sanguíneo de Kell/genética , FenótipoRESUMO
Ten cattle farms located in an area with a recent history of poultry botulism outbreaks were investigated to evaluate the occurrence of toxigenic C. botulinum in healthy cattle. Environmental samples in the 10 cattle farms and bovine fecal contents in farms with a confirmed environmental contamination were collected. Detection of C. botulinum toxin genes C, D, C/D, D/C and E was performed using real-time PCR. 4.9% (7/143) of the environmental samples collected in the 10 investigated cattle farms were positive for C. botulinum type C/D. Theses samples (boot-swabs in stalls and on pasture and water of a stream) were collected in 3 different farms. One cow dung sample and 3 out of 64 fecal contents samples collected in a single farm were also positive for C. botulinum type C/D. This study demonstrates that cattle are probably indirectly contaminated via poultry botulism in the area and that they can be intermittent carrier of C. botulinum type C/D after poultry botulism outbreaks in mixed farms.
Assuntos
Botulismo/veterinária , Clostridium botulinum/isolamento & purificação , Surtos de Doenças/veterinária , Microbiologia Ambiental , Doenças das Aves Domésticas/microbiologia , Animais , Botulismo/epidemiologia , Botulismo/microbiologia , Bovinos , Fezes/microbiologia , Feminino , Aves Domésticas , Doenças das Aves Domésticas/epidemiologiaRESUMO
Between 2011 and 2013, 17 poultry botulism outbreaks were investigated in France. All cases were associated with Clostridium botulinum type C-D. Presence of C. botulinum was studied in seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoirs, water, and feed. Swabs, litter, soil, darkling beetles, rodents and wild bird droppings, feed and water samples were collected. The presence of C. botulinum type C-D in the environment of affected flocks was detected in 39.5% of the 185 samples analysed by real-time polymerase chain reaction. C. botulinum type C-D was reported in each area. Four areas were more frequently contaminated, being found positive in more than one-half of farms: darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). After cleaning and disinfection, the ventilation system and/or the soil (in the houses and the surroundings) returned positive results in four out of eight poultry farms. Consequently, darkling beetles, the drinking water, the ventilation system and the soil in the surroundings and the houses were identified as the main critical contaminated areas to consider in poultry farms to prevent recurrence of botulism outbreaks.
Assuntos
Botulismo/veterinária , Clostridium botulinum/isolamento & purificação , Abrigo para Animais/normas , Doenças das Aves Domésticas/microbiologia , Aves Domésticas , Animais , Botulismo/epidemiologia , Botulismo/microbiologia , Surtos de Doenças/veterinária , Microbiologia Ambiental , Feminino , França/epidemiologia , Masculino , Doenças das Aves Domésticas/epidemiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
1. The growth of Bacillus cereus group bacteria often limits the shelf-life of pasteurised liquid egg products and is also a putative toxin producer. This study was performed to better understand the route of contamination by B. cereus in egg products by studying the factors affecting eggshell contamination on-farm. 2. Eggs were collected in warm and cold seasons in 50 conventional laying farms in Western France. Egg surfaces were analysed for the presence of B. cereus group bacteria, environmental measurements were recorded and production practices were identified through a questionnaire filled out by the farmers. 3. A total of 44% of the farms were contaminated by mesophilic and 10% by psychrotrophic B. cereus group bacteria. No significant effect of the season was observed, whatever the thermal type. Several procedures were associated with reduced eggshell contamination by mesophilic bacteria, including the limitation of dust formation from manure and feeding and efficient disinfection of the silo, houses and the sanitary wall between houses. 4. The research highlights the need to promote prevention strategies, from farm to fork, for the control of eggshell and putative subsequent egg product contamination by B. cereus group bacteria.
Assuntos
Bacillus cereus/isolamento & purificação , Galinhas/microbiologia , Casca de Ovo/microbiologia , Criação de Animais Domésticos/métodos , Animais , Desinfecção/métodos , Feminino , Microbiologia de Alimentos , França , Abrigo para Animais , Estações do AnoRESUMO
The aim of the present study was (i) to type, by genotypic and phenotypic methods, a collection of psychrotrophic bacteria belonging to the Bacillus cereus group collected in a farm and in 6 egg breaking industries during a period covering a warm and a cold season, and (ii) to characterize the egg product spoilage (growth in liquid whole egg) and the sanitary risk potential (cytotoxic activity on Caco-2 cells and adhesion on stainless steel) of each isolate of the collection. The investigation of specific psychrotrophic and mesophilic signatures together with the study of ability to grow at 6 °C and/or at 43 °C on optimal agar medium allowed highlighting twelve profiles, the major one corresponding to the species Bacillus weihenstephanensis (46.2% of the collection). The diversity of the profiles depended on the season and on the origin of the isolates. In terms of food spoilage, all the isolates were able to grow at the same level in liquid whole egg and in optimal medium, even at low temperature. Under the same conditions, the cytotoxic activity depended on the isolate, the medium and the temperature. At 10 °C, no isolate was cytotoxic at 10 °C in liquid whole egg and only one, belonging to the Bacillus weihenstephansensis species, in the optimal medium. All the isolates were able to adhere on stainless steel at various levels, from 2.6±0.2 log cfu/cm(2) to 4.9±0.1 log cfu/cm(2). A large majority (80.8%) was strongly adhering and could lead to the formation of biofilms in industrial equipments.
Assuntos
Bacillus cereus , Aderência Bacteriana/fisiologia , Qualidade de Produtos para o Consumidor , Ovos/microbiologia , Contaminação de Alimentos/análise , Bacillus cereus/classificação , Bacillus cereus/isolamento & purificação , Bacillus cereus/fisiologia , Biodiversidade , Microbiologia de Alimentos , Humanos , Filogenia , Medição de Risco , Estações do Ano , Aço InoxidávelRESUMO
Eleven affected members of a large German-American family segregating recessively inherited, congenital, non-syndromic sensorineural hearing loss (SNHL) were found to be homozygous for the common 35delG mutation of GJB2, the gene encoding the gap junction protein Connexin 26. Surprisingly, four additional family members with bilateral profound SNHL carried only a single 35delG mutation. Previously, we demonstrated reduced expression of both GJB2 and GJB6 mRNA from the allele carried in trans with that bearing the 35delG mutation in these four persons. Using array comparative genome hybridization (array CGH), we have now identified on this allele a deletion of 131.4 kb whose proximal breakpoint lies more than 100 kb upstream of the transcriptional start sites of GJB2 and GJB6. This deletion, del(chr13:19,837,344-19,968,698), segregates as a completely penetrant DFNB1 allele in this family. It is not present in 528 persons with SNHL and monoallelic mutation of GJB2 or GJB6, and we have not identified any other candidate pathogenic copy number variation by arrayCGH in a subset of 10 such persons. Characterization of distant GJB2/GJB6 cis-regulatory regions evidenced by this allele may be required to find the 'missing' DFNB1 mutations that are believed to exist.
Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência , Alelos , Sequência de Bases , Deleção Cromossômica , Cromossomos Humanos Par 13/genética , Hibridização Genômica Comparativa , Conexina 26 , Conexina 30 , Saúde da Família , Feminino , Testes Genéticos , Genótipo , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Penetrância , Homologia de Sequência do Ácido NucleicoRESUMO
Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/análise , Mastite/veterinária , Proteoma/análise , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Animais , Técnicas Bacteriológicas , Contagem de Colônia Microbiana , Meios de Cultura/química , Feminino , Mastite/imunologia , Ovinos , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine hosts regardless the locus of isolation (nares and udder). By pulsed-field gel electrophoresis, seven major pulsotypes were identified among 153 isolates recovered from 12 different regions of France as well as from Brazil, the USA and Belgium. Typing of the accessory gene regulator (agr) and capsular (cap) serotype was carried out on all the isolates and revealed the predominance of agr I and III and of cap8 regardless the ruminant host species. Screening for methicilin-resistant S. aureus (MRSA) was carried out by disk diffusion and revealed a prevalence of only 3.2% of MRSA among the strains tested. These results suggest the existence of a host rather than tissue specificity among S. aureus isolates colonising the ruminant species and suggest a limited transmission of those isolates between large (bovine) and small (ovine-caprine) ruminants. The agr class and cap types correlated with pulsotype clusters rather than with a specific host species. Antimicrobial resistance appears not to have contributed to the predominance of any given genotypes, and MRSA prevalence appears very low in ruminant isolates.
Assuntos
Doenças dos Bovinos/microbiologia , Doenças das Cabras/microbiologia , Doenças dos Ovinos/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/classificação , Animais , Cápsulas Bacterianas/metabolismo , Bovinos , Regulação Bacteriana da Expressão Gênica , Genótipo , Cabras , Interações Hospedeiro-Patógeno , Filogenia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Ovinos , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND AND AIMS: The prevalence and natural history of hereditary pancreatitis (HP) remain poorly documented. The aims of this study were to assess genetic, epidemiological, clinical and morphological characteristics of HP in an extensive national survey. METHODS: A cohort comprising all HP patients was constituted by contacting all gastroenterologists and paediatricians (response rate 84%) and genetics laboratories (response rate 100%) in France (60,200,000 inhabitants). Inclusion criteria were the presence of mutation in the cationic trypsingen gene (PRSS1 gene), or chronic pancreatitis in at least two first-degree relatives, or three second-degree relatives, in the absence of precipitating factors for pancreatitis. RESULTS: 78 families and 200 patients were included (181 alive, 6673 person-years, males 53%, alcoholism 5%, smoking 34%). The prevalence was 0.3/100,000 inhabitants. PRSS1 mutations were detected in 68% (R122H 78%, N29I 12%, others 10%). Penetrance was 93%. Median age at first symptom, diagnosis and date of last news, were 10 (range 1-73), 19 (1-80) and 30 (1-84) years, respectively. HP was responsible for pancreatic pain (83%), acute pancreatitis (69%), pseudocysts (23%), cholestasis (3%), pancreatic calcifications (61%), exocrine pancreatic insufficiency (34%, median age of occurrence 29 years), diabetes mellitus (26%, median age of occurrence 38 years) and pancreatic adenocarcinoma (5%, median age 55 years). No differences in clinical and morphological data according to genetic status were observed. 19 patients died, including 10 directly from HP (8 from pancreatic adenocarcinoma). CONCLUSION: The prevalence of HP in France is at least 0.3/100,000. PRSS1 gene mutations are found in 2/3 with a 93% penetrance. Mutation type is not correlated with clinical/morphological expression. Pancreatic adenocarcinoma is the cause of nearly half the deaths.
Assuntos
Pancreatite Crônica/genética , Adenocarcinoma/epidemiologia , Adenocarcinoma/etiologia , Adolescente , Adulto , Distribuição por Idade , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Métodos Epidemiológicos , Insuficiência Pancreática Exócrina/epidemiologia , Insuficiência Pancreática Exócrina/etiologia , Feminino , França/epidemiologia , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/etiologia , Pancreatite Crônica/complicações , Pancreatite Crônica/epidemiologia , Penetrância , Fenótipo , Tripsina , Tripsinogênio/genética , Adulto JovemRESUMO
AIMS: To report on a family with five members who carry the A3243G mutation in mitochondrial tRNA for leucine 1 (MTTL1) and present with diabetes, chronic intestinal pseudo-obstruction (CIPO) and recurrent pancreatitis, and to screen for this mutation in a cohort of 36 unrelated patients with recurrent pancreatitis. METHODS: The mutation was quantified in several tissue samples from patients. Respiratory chain activity was studied in muscle biopsies and fibroblast cultures. In addition, the thymidine phosphorylase gene (TP) involved in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and three genes involved in chronic pancreatitis - PRSS1, SPINK1 and CFTR - were sequenced in affected patients. Finally, the MTTL1 gene was examined in 36 unrelated patients who had recurrent pancreatitis, but no mutations in the PRSS1 and SPINK1 genes. RESULTS: Heteroplasmy for the mtDNA A3243G mutation was found in all tissue samples from these patients, but no mutations were found in the genes coding for thymidine phosphorylase, PRSS1, SPINK1 and CFTR. Also, none of the 36 unrelated patients with recurrent pancreatitis were carrying any MTTL1 mutations. CONCLUSION: The mtDNA A3243G mutation associated with the gastrointestinal manifestations observed in the affected family should be regarded as a possible cause of CIPO and unexplained recurrent pancreatitis. However, the mutation is probably only weakly involved in cases of isolated recurrent pancreatitis.
Assuntos
DNA Mitocondrial/genética , Complicações do Diabetes/genética , Diabetes Mellitus/genética , Pseudo-Obstrução Intestinal/genética , Pancreatite/genética , Polimorfismo de Nucleotídeo Único , Surdez/genética , Complicações do Diabetes/patologia , Diabetes Mellitus/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Mutação , Linhagem , RecidivaRESUMO
In 1996, shortly after a locus for hereditary pancreatitis had been mapped to chromosome 7q35, an apparent gain-of-function missense mutation, p.R122H, in the cationic trypsinogen gene (PRSS1) was identified. Thereafter, the search for chronic pancreatitis-associated genetic factors has been largely focused on one form of genetic variation, namely, single nucleotide substitutions (SNSs). Only very recently has another type of genetic variation - copy number variations (CNVs) - been found to cause the disease. First, we identified duplication and triplication of an approximately 605 kb segment on chromosome 7q35 in French white patients with hereditary or idiopathic chronic pancreatitis. These alterations increased the copy number of PRSS1 as well as PRSS2, which encodes anionic trypsinogen. Second, we characterized a hybrid trypsinogen gene, in which exons 1 and 2 were derived from PRSS2 and exons 3 to 5 from PRSS1. Interestingly, this hybrid gene had two independent gain-of-function effects: increased trypsinogen gene copy number and it contained the p.N29I pancreatitis-causing missense mutation. Lastly, we identified two loss-of-function copy number mutations (deletions) in the SPINK1 gene, which encodes pancreatic secretory trypsin inhibitor (PSTI). Particularly, in one family with chronic pancreatitis, deletion of the complete SPINK1 gene was co-inherited with a CFTR missense mutation (p.L997F), revealing another layer of complexity between CNV and SNS interactions in the determination of a given disease phenotype. These findings represent a further demonstration of how studies of CNVs have altered the landscape of genetic research in the past few years and offer a fresh glimpse into the exciting realm of human CNVs.
Assuntos
Dosagem de Genes/genética , Pancreatite Crônica/genética , Duplicação Gênica , Genoma/genética , Humanos , Mutação/genética , Pancreatite Crônica/classificação , Tripsinogênio/genéticaRESUMO
BACKGROUND: Previous studies have reported conflicting results regarding recurrent pregnancy loss and skewed X-chromosome inactivation. Hence, we sought an association by carrying out a specifically designed incident paired case-control study with required statistical power. METHODS: Design incident 1:3 matched case-control study, from 2003 to 2007. SETTING: University Hospital of Brest. PATIENTS: Women, from the Brittany area, consecutively referred for at least two unexplained consecutive spontaneous abortions. CONTROLS: Women from the same geographic area, with no history of pregnancy loss and at least one normal pregnancy, recruited using electoral lists and then paired with cases, with respect to age, to within 1 year. INTERVENTION: Assessment of skewed X-chromosome inactivation. STATISTICAL ANALYSIS: Comparison of the ratio of >90% skewed X-chromosome inactivation by conditional logistic regression. RESULTS: Five hundred and forty-three controls (mean age: 34.3 years) were paired within 1 year to 200 cases. The cases (mean age: 33.6 years) had experienced between 2 and 14 consecutive losses (median 3). The rate of >90% skewed X-chromosome inactivation was not statistically different (P = 0.33, odds ratio: 0.58, 95% confidence interval: 0.19-1.77) between cases and paired controls, 2.27% versus 4.1%, respectively. CONCLUSIONS: We conclude that there is no association between skewed X-chromosome inactivation and recurrent pregnancy loss, defined as two or more unexplained consecutive spontaneous abortions.
Assuntos
Aborto Habitual/diagnóstico , Aborto Habitual/etiologia , Aberrações Cromossômicas , Inativação do Cromossomo X , Aborto Espontâneo/diagnóstico , Aborto Espontâneo/etiologia , Adolescente , Adulto , Estudos de Casos e Controles , Cromossomos Humanos X , Feminino , Humanos , Modelos Estatísticos , Gravidez , Projetos de PesquisaRESUMO
Mutations in the serine protease inhibitor Kazal type 1 gene (SPINK1) encoding pancreatic secretory trypsin inhibitor (PSTI) have recently been found to be associated with chronic pancreatitis. Nevertheless, knowledge of severe mutations is particularly scarce, both in terms of number and in the extent of clinical information. The aim of this study was to expand the known spectrum of such mutations. 46 unrelated families, each including at least two pancreatitis patients and carrying neither cationic trypsinogen (PRSS1) mutations nor the frequent SPINK1 N34S mutation, participated in this study. The four exons and their flanking sequences of the SPINK1 gene were screened by denaturing high performance liquid chromatography analysis (DHPLC); and mutations were identified by direct sequencing. A heterozygous microdeletion mutation (c.27delC), which occurs within a symmetric element, was identified in two families. In one family, c.27delC showed segregation with the disease across two generations, with a penetrance of up to 75%. But in the other family, however, the same mutation manifested as a low-penetrance susceptibility factor. In addition, a novel heterozygous splicing mutation, c.87+1G>A (G>A substitution at nucleotide +1 of intron 2) was found in one family with familial pancreatitis. Our results also helped to resolve the sharply differing views about PSTI's role in pancreatitis.
Assuntos
Mutação/genética , Pancreatite/genética , Inibidor da Tripsina Pancreática de Kazal/genética , Adulto , Criança , Estudos de Coortes , Éxons/genética , Feminino , Deleção de Genes , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Sítios de Splice de RNA/genéticaRESUMO
BACKGROUND: R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365 through 366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. MATERIALS AND METHODS: DNA samples containing either the known c.365G>A or c.365 through 366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay. RESULTS: DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C). CONCLUSIONS: A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Análise Mutacional de DNA/métodos , Pancreatite/genética , Tripsina , Tripsinogênio/genética , Substituição de Aminoácidos , DNA/química , Conversão Gênica , Predisposição Genética para Doença , Humanos , Mutação de Sentido Incorreto , Desnaturação de Ácido Nucleico , Tripsinogênio/química , Tripsinogênio/metabolismoRESUMO
Over the past 5 years, several gain-of-function missense mutations in the human cationic trypsinogen gene (PRSS1, OMIM 276000) have been associated with hereditary and/or sporadic pancreatitis. This study reports a new pancreatitis-associated mutation--R116C (CGT > TGT: c.346C > T)--in the gene.