RESUMO
Objective: Familial hypercholesterolaemia (FH) variant positive subjects have over double the cardiovascular risk of low-density-lipoprotein-cholesterol (LDL-C) matched controls. It is desirable to optimise FH variant detection. Methods: We identified 213 subjects with FH gene panel reports (LDLR, APOB, PCSK9, and APOE) based on total cholesterol >310 mg/dL; excluding triglycerides >400 mg/dL, cascade screening, and patients without pre-treatment LDL-C recorded. Demographic, clinical and lipid parameters were recorded. Results: A 31/213 (14.6%) patients had pathogenic or likely pathogenic FH variants. 10/213 (4.7%) had variants of uncertain significance. Compared with patients without FH variants, patients with FH variants were younger (median age, 39 years vs. 48 years), had more tendon xanthomata (25.0% vs. 11.4%), greater proportion of first degree relatives with total cholesterol >95th percentile (40.6% vs. 16.5%), higher LDL-C (median, 271 mg/dL vs. 236 mg/dL), and lower triglycerides (median, 115 mg/dL vs. 159 mg/dL). The Besseling et al. model (c-statistic 0.798) improved FH variant discrimination over Friedewald LDL-C (c-statistic 0.724), however, Dutch Lipid Clinic Network Score (DLCNS) did not (c-statistic 0.665). Sampson LDL-C (c-statistic 0.734) had similar discrimination to Friedewald. Conclusion: Although tendon xanthomata and first degree relatives with high total cholesterol >95th percentile were associated with FH variants, DLCNS or Simon Broome criteria did not improve FH detection over LDL-C. Sampson LDL-C did not significantly improve discrimination over Friedewald. Although lower triglycerides and younger age of presentation are positively associated with presence of FH variants, this information is not commonly used in FH detection algorithms apart from Besseling et al.
RESUMO
BACKGROUND: Type I hyperlipoproteinemia, manifesting as chylomicronemia and severe hypertriglyceridemia, is a rare autosomal recessive disorder usually caused by mutations in the lipoprotein lipase gene (LPL). OBJECTIVE: We sought to determine whether mutations in LPL could explain the clinical indications of a patient presenting with pancreatitis and hypertriglyceridemia. METHODS: Coding regions of LPL were amplified by polymerase chain reaction and analyzed by nucleotide sequencing. The LPL messenger RNA transcript was also analyzed to investigate whether alternative splicing was occurring. RESULTS: The patient was homozygous for the mutation c.767_768insTAAATATT in exon 5 of the LPL gene. This mutation is predicted to result in either a truncated nonfunctional LPL, or alternatively a new 5' donor splice site may be used, resulting in a full-length LPL with an in-frame deletion of 3 amino acids. Analysis of messenger RNA from the patient showed that the new splice site is used in vivo. CONCLUSION: Homozygosity for a mutation in the LPL gene was consistent with the clinical findings. Use of the new splice site created by the insertion mutation rescues an otherwise damaging frameshift mutation, resulting in expression of an almost full-length LPL that is predicted to be partially functional. The patient therefore has a less severe form of type I hyperlipoproteinemia than would be expected if she lacked any functional LPL.
Assuntos
Mutação da Fase de Leitura , Lipase Lipoproteica/genética , Splicing de RNA , Adulto , Sequência de Bases , Éxons/genética , Feminino , Homozigoto , Humanos , Hiperlipoproteinemias/enzimologia , Hiperlipoproteinemias/genética , Mutagênese Insercional , RNA Mensageiro/genéticaRESUMO
The genus Pseudomonas contains fast-growing nutritionally versatile bacteria that are able to utilize a wide variety of carbon sources. The ubiquity of the genus has been highlighted by conventional microbiology and the genus is well represented in collections of cultured bacteria. Here we evaluate the Pseudomonas population in New Zealand soils by comparing a culture-independent (real-time PCR combined with fluorescent TaqMan technology) with a culture-dependent (Gould's S1) population estimate. We show that cultivated fluorescent pseudomonads are not numerically dominant and represent a small proportion of <1% of the total Pseudomonas population, and that the total Pseudomonas population itself represents only a small proportion of <1% of the total bacterial population.
Assuntos
Reação em Cadeia da Polimerase/métodos , Pseudomonas/isolamento & purificação , Técnicas Bacteriológicas/métodos , Contagem de Colônia Microbiana , DNA Bacteriano/química , DNA Bacteriano/genética , Nova Zelândia , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Microbiologia do Solo , Taq Polimerase/metabolismoRESUMO
The aim of the study was to determine the molecular cause of dysfibrinogenaemia in a woman with a prolonged thrombin time. Functional fibrinogen abnormalities can be benign or may lead to bleeding or thrombotic conditions. In complex cases, phenotypes may be acquired or involve interplay between several coinherited mutations. The authors developed a new whole-protein time-of-flight mass spectrometry (TOF MS) approach to direct targeted DNA sequencing of the fibrinogen genes and determine the phase of multiple substitutions in a single individual. TOF MS analysis of the individual's fibrinogen indicated normal Bß, γ, and alternately transcribed γ' chain isoforms, but aberrant Aα chain masses. Subsequent fibrinogen Aα gene (FGA) sequencing indicated the presence of three different mutations. Two of the substitutions, Aα17GlyâCys (at the thrombin cleavage site) and Aα381SerâPhe (in the αC connector) were novel and the third, Aα312ThrâAla, was a known benign polymorphism. Accurate mass measurements of isolated control Aα chains showed the predicted Aα polypeptide at 66â132âDa and additional phosphorylated species at +â80 and +â160âDa. Patient's Aα chains on the other hand had masses of 66â103 and 66â241âDa indicating that she had one 312Ala allele (-30âDa) and one 312Thr allele which carried both the 17GlyâCys (+â46âDa) and 381SerâPhe (+â60)âDa mutations. Cotransmission of these new mutations was confirmed by Aα chain TOF MS of plasma fibrinogen and targeted FGA nucleotide sequencing for 10 additional family members.
Assuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Mutação , Adulto , Afibrinogenemia/etnologia , Afibrinogenemia/patologia , Alelos , Substituição de Aminoácidos , Povo Asiático , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tempo de TrombinaAssuntos
Fibrinogênio/genética , Fibrinogênio/metabolismo , Mutação , Peptídeos/química , Adolescente , Afibrinogenemia/sangue , Alelos , Motivos de Aminoácidos , Carboxipeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Hepatócitos/citologia , Humanos , Hidrólise , Masculino , Fosforilação , Pré-Albumina/química , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização ProteicaAssuntos
Afibrinogenemia/genética , Coagulação Sanguínea/genética , Fibrinogênio/genética , Genótipo , Mutação/genética , Pré-Escolar , Pai , Fibrinogênio/biossíntese , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Espectrometria de Massas , Linhagem , Polimorfismo GenéticoAssuntos
Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Afibrinogenemia/sangue , Substituição de Aminoácidos , Códon , Fibrinogênios Anormais/química , Fibrinogênios Anormais/metabolismo , Expressão Gênica , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Fibrinogênio/genética , Fibrinogênios Anormais/genética , Mutação , Afibrinogenemia/sangue , Afibrinogenemia/genética , Sequência de Aminoácidos , Éxons , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibrinogênios Anormais/química , Fibrinogênios Anormais/metabolismo , Humanos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Multimerização Proteica/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Sítios de Splice de RNA , Deleção de Sequência , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Mutação Puntual , Adulto , Cesárea , Feminino , Fibrinogênio/análise , Humanos , Gravidez , Adulto JovemRESUMO
AIM: To identify the diagnostic and treatment rates for familial hypercholesterolaemia (FH) in New Zealand. METHODS: The FH data held by Canterbury Health Laboratories and the Canterbury District Health Board lipid clinic was examined to give an indication of the level of identification and treatment of FH in both Canterbury and New Zealand. RESULTS: Between 2004-08, 588 people, out of a possible 10,500 affected people, who presented with a pre-treatment cholesterol =8.0 mmol/L, lipid stigmata or a strong family history of cardiovascular disease (CVD), were tested for low density lipoprotein (LDLR) and apolipoprotein B (APOB) mutations. Mutations were identified in 76 cases (13%). 353 relatives were screened and 159 (45%) were found to have FH. This data suggests that less than 20% of the affected people in Canterbury have been diagnosed and less than 2.2% nationally. CONCLUSION: FH diagnostic services in New Zealand appear significantly underdeveloped thereby denying affected people the opportunity of early treatment to reduce the risk of premature cardiovascular events. Cascade screening is shown to be a cost effective and efficient approach to identifying people with FH.
Assuntos
Doenças Cardiovasculares/genética , Doenças Cardiovasculares/prevenção & controle , Hipercolesterolemia/epidemiologia , Hipercolesterolemia/genética , Doenças Cardiovasculares/epidemiologia , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/terapia , Programas de Rastreamento , Mutação , Nova Zelândia/epidemiologia , Fatores de RiscoRESUMO
OBJECTIVES: High-resolution melting analysis has been validated for screening several genes of clinical significance. We evaluated whether it would be useful for detecting mutations in the LDL receptor gene (LDLR). DESIGN AND METHODS: We analysed 60 samples heterozygous for different LDLR sequence variants, and compared the melting curves to those of the corresponding homozygous normal amplicons to assess whether they would be detected in a mutation scanning screen using melting analysis. RESULTS: Although six mutations in exon 4 did not yield usable melting data, 96% (52/54) of the other variants were detected by the melting analysis, while two variants were poorly resolved. CONCLUSIONS: Melting analysis is a rapid and sensitive mutation scanning technique, but is not suitable for some GC-rich amplicons such as LDLR exon 4, or exons with high frequencies of benign polymorphisms which would require follow-up DNA sequencing. We developed a successful strategy which combines melting analysis with direct sequencing of problematic amplicons.
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DNA/análise , Testes Genéticos/métodos , Desnaturação de Ácido Nucleico , Receptores de LDL/genética , Cromatografia Líquida de Alta Pressão , Variação Genética , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Genético , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Receptores de LDL/análise , Sensibilidade e Especificidade , Análise de Sequência de DNA , Temperatura de TransiçãoRESUMO
We analysed the Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) exons and intronic junctions of 71 patients with familial hypercholesterolemia (FH) in whom LDL receptor (LDLR) or apolipoprotein B100 mutations were excluded. The previously reported S127R and R237W mutations were found in South African families, whereas new missense mutations D129G and A168E were found in families from New Zealand. Only, the S127R and D129G mutations modify a highly conserved residue and segregate with the FH phenotype. We overexpressed those mutants in hepatoma cells and found that both S127R and D129G have reduced autocatalytic activity compared with wild-type PCSK9, whereas the A168E mutant is processed normally. The S127R and D129G mutants were not secreted from cells, unlike the A168E mutant and wild-type PCSK9. By immunoblot, we showed that the expression of the LDLR was reduced by 40% in cells overexpressing wild-type or A168E PCSK9 and further reduced by 30% when the S127R or D129G mutants were used. Paralleling the LDLR levels, LDL cellular binding decreased by 25% upon wild-type PCSK9 or A168E overexpression, and by 45% with both S127R and D129G mutants. Our study therefore indicates that PCSK9 mediated inhibition of the LDLR does not require PCSK9 autocatalytic cleavage or secretion, suggesting that PCSK9 may also function intracellularly.
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Hiperlipoproteinemia Tipo II/genética , Serina Endopeptidases/genética , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apolipoproteína B-100/genética , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Nova Zelândia , Linhagem , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/genética , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , África do SulRESUMO
BACKGROUND: Single base-pair substitution mutations in the gene for coagulation factor VIII, procoagulant component (hemophilia A) (F8) account for approximately 50% of severe cases of hemophilia A (HA), and almost all moderate or mild cases. Because F8 is a large gene, mutation screening using denaturing HPLC or DNA sequencing is time-consuming and expensive. METHODS: We evaluated high-resolution melting analysis as an option for screening for F8 gene mutations. The melting curves of amplicons heterozygous for known F8 gene mutations were compared with melting curves of the corresponding normal amplicons to assess whether melting analysis could detect these variants. We examined 2 platforms, the Roche LightCycler 480 (LC480) and the Idaho Technology LightScanner. RESULTS: On both instruments, 18 (90%) of the 20 F8 gene variants we examined were resolved by melting analysis. For the other 2 mutations, the melting curves of the heterozygous amplicons were similar to the corresponding normal amplicons, suggesting these variants may not be detected by this approach in a mutation-scanning screen. CONCLUSION: High-resolution melting analysis is an appealing technology for F8 gene screening. It is rapid and quickly identifies mutations in the majority of HA patients; samples in which no mutation is detected require further testing by DNA sequencing. The LC480 and LightScanner platforms performed similarly.
Assuntos
Fator VIII/genética , Hemofilia A/genética , Humanos , Masculino , Mutação , Temperatura de TransiçãoAssuntos
Remoção de Componentes Sanguíneos/métodos , LDL-Colesterol/sangue , Hiperlipoproteinemia Tipo II/terapia , Adolescente , Anticolesterolemiantes/administração & dosagem , Azetidinas/administração & dosagem , LDL-Colesterol/efeitos dos fármacos , Quimioterapia Combinada , Ezetimiba , Feminino , Fluorbenzenos/administração & dosagem , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Pirimidinas/administração & dosagem , Rosuvastatina Cálcica , Sulfonamidas/administração & dosagemRESUMO
Some promoters, including the DmpR-controlled sigma(N)-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping sigma(D)-factor can restore sigma(N)-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivo transcription competition assays, we show that all the four sigma(D) mutant proteins are defective in their ability to compete with sigma(N) for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of sigma(D) or overproduction of the anti-sigma(D) protein Rsd were also found to allow (p)ppGpp-independent transcription from the sigma(N)-Po promoter. Together with data from the direct effects of (p)ppGpp on sigma(N)-dependent Po transcription and sigma-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative sigma-factor competition to adapt to changing cellular nutritional demands.