Association of Clinical Characteristics With Familial Hypercholesterolaemia Variants in a Lipid Clinic Setting: A Case-Control Study.

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.

A novel frameshift mutation in the lipoprotein lipase gene is rescued by alternative messenger RNA splicing.

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.

Novel fibrinogen mutations (Aα17Gly→Cys and Aα381Ser→Phe) occurring with a 312Thr→Ala polymorphism: allelic phase assigned by direct mass measurement.

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, Aα17Gly→Cys (at the thrombin cleavage site) and Aα381Ser→Phe (in the αC connector) were novel and the third, Aα312Thr→Ala, was a known benign polymorphism. Accurate mass measurements of isolated control Aα chains showed the predicted Aα polypeptide at 66 132 Da and additional phosphorylated species at + 80 and + 160 Da. Patient's Aα chains on the other hand had masses of 66 103 and 66 241 Da indicating that she had one 312Ala allele (-30 Da) and one 312Thr allele which carried both the 17Gly→Cys (+ 46 Da) and 381Ser→Phe (+ 60) Da 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.

Preventing cardiovascular disease: a review of the effectiveness of identifying the people with familial hypercholesterolaemia in New Zealand.

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.

Evaluation of high-resolution melting analysis for screening the LDL receptor gene.

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.

Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa.

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.

Detection of factor VIII gene mutations by high-resolution melting analysis.

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.

Low density lipoprotein--receptor (LDL-R) gene mutations among Filipinos with familial hypercholesterolemia.

BACKGROUND: Familial Hypercholesterolemia (FH) is an autosomal dominant disease resulting from mutations of the LDL (LDLR) receptor gene leading to a diminished catabolism and elevated level of LDL cholesterol (LDL-C). It is associated with an increased risk for cardiovascular disease (CVD). The MEDPED (Make Early Diagnosis-Prevent Early Death) program, an initiative cited by the WHO Human Genetics Programme in their report on FH, initiated international collaboration to identify and follow-up patients with FH globally. From Asia-Pacific, only 6 countries are participating and no data among Filipinos particularly on genetic profiles is available at present. This study attempts to initiate data collection and participation in the global initiative. OBJECTIVES: Primary: 1. To describe the phenotype of Filipino patients with FH. 2. To determine and characterize the LDL-R gene mutations among Filipino patients with clinical features of FH. Secondary: To determine the association of the clinical characteristics of FH with the presence of LDLR gene mutations. DESIGN: Cross- Sectional Study. SETTING: Multicenter, Outpatient Clinic. PARTICIPANTS: 60 unrelated patients, 18 y/o and above from UP-PGH, Manila Doctors Hospital and Cardinal Santos Medical Center. FH was diagnosed according to the Dutch Lipid Clinic Network Criteria cited by WHO which is based on a history of premature CVD, family history, tendon xanthoma, arcus cornealis, and LDL C levels. METHODS: With informed consent, clinical history, physical examination and lipid profile data were determined. Blood samples were extracted, processed to isolate DNA specimens at the National Institutes of Health, Institute of Human Genetics, and sent to Canterbury Health Laboratories at Christchurch, New Zealand for DNA analysis. ANALYSIS: Descriptive statistics, Fisher's exact test and Student's t-test using Stata version 6.0 software. RESULTS: Sixty patients with a mean age of 55 y/o were included, including 39 (65%) females. The mean LDL level was 227 mg/dl. Cardiovascular Disease and a family history of dyslipidemia were present in 55 & 60% of the samples, respectively. Twenty percent had documented LDL-R gene mutations. Six of the mutations were considered novel. A family history of dyslipidemia, an elevated LDL-C level, and a high FH score exhibited a statistically significant association with mutations. The study population has a high prevalence of CVD at an average age of 55 years with a strong family history of dyslipidemia and very high average LDL-C levels. One out of every 5 patients had LDL-R gene mutations, 6 of which were considered novel. LDL-R gene mutation was significantly associated with family history of dyslipidemia, LDL-C Level and FH score. CLINICAL AND RESEARCH IMPLICATION: This is the first international collaborative genetic study among Filipinos with FH. Data could allow the country to participate in the WHO/MEDPED global program. Collaborative efforts will lead to more effective detection, treatment and prevention of CV events. Novel mutations were discovered and further analysis of these genes will be done.

Quantification of the Pseudomonas population in New Zealand soils by fluorogenic PCR assay and culturing techniques.

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.

The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.

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.