Genetic Testing of Korean Familial Hypercholesterolemia Using Whole-Exome Sequencing

Familial hypercholesterolemia (FH) is a genetic disorder with an increased risk of early-onset coronary artery disease. Although some clinically diagnosed FH cases are caused by mutations in LDLR, APOB, or PCSK9, mutation detection rates and profiles can vary across ethnic groups. In this study, we aimed to provide insight into the spectrum of FH-causing mutations in Koreans. Among 136 patients referred for FH, 69 who met Simon Broome criteria with definite family history were enrolled. By whole-exome sequencing (WES) analysis, we confirmed that the 3 known FH-related genes accounted for genetic causes in 23 patients (33.3%). A substantial portion of the mutations (19 of 23 patients, 82.6%) resulted from 17 mutations and 2 copy number deletions in LDLR gene. Two mutations each in the APOB and PCSK9 genes were verified. Of these anomalies, two frameshift deletions in LDLR and one mutation in PCSK9 were identified as novel causative mutations. In particular, one novel mutation and copy number deletion were validated by co-segregation in their relatives. This study confirmed the utility of genetic diagnosis of FH through WES.     

Genetic Polymorphisms in LDLR,APOB, PCSK9 and Other Lipid Related Genes Associated with Familial Hypercholesterolemia in Malaysia

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevations in total cholesterol (TC) and low density lipoprotein cholesterol (LDLc). Development of FH can result in the increase of risk for premature cardiovascular diseases (CVD). FH is primarily caused by genetic variations in Low Density Lipoprotein Receptor (LDLR), Apolipoprotein B (APOB) or Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) genes. Although FH has been extensively studied in the Caucasian population, there are limited reports of FH mutations in the Asian population. We investigated the association of previously reported genetic variants that are involved in lipid regulation in our study cohort. A total of 1536 polymorphisms previously implicated in FH were evaluated in 141 consecutive patients with clinical FH (defined by the Dutch Lipid Clinic Network criteria) and 111 unrelated control subjects without FH using high throughput microarray genotyping platform. Fourteen Single Nucleotide Polymorphisms (SNPs) were found to be significantly associated with FH, eleven with increased FH risk and three with decreased FH risk. Of the eleven SNPs associated with an increased risk of FH, only one SNP was found in the LDLR gene, seven in the APOB gene and three in the PCSK9 gene. SNP rs12720762 in APOB gene is associated with the highest risk of FH (odds ratio 14.78, p<0.001). Amongst the FH cases, 108 out of 141 (76.60%) have had at least one significant risk-associated SNP. Our study adds new information and knowledge on the genetic polymorphisms amongst Asians with FH, which may serve as potential markers in risk prediction and disease management.     

New species of Stenandrium (Acanthaceae) from the Planalto of Brazil

Three new species ofStenandrium,S. hatschbachii from Minas Gerais,S. goiasense from Goiás, andS. irwinii also from Goiás, are described, illustrated, and compared with their closest relatives.     

Common mutations of familial hypercholesterolemia patients in Taiwan: characteristics and implications of migrations from southeast China

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China.     

Genetic Variants of LDLR and PCSK9 Associated with Variations in Response to Antihypercholesterolemic Effects of Armolipid Plus with Berberine

Background

Armolipid Plus (AP) is a nutraceutical that contains policosanol, fermented rice with red yeast, berberine, coenzyme Q10, folic acid, and astaxanthin. It has been shown to be effective in reducing plasma LDL cholesterol (LDLc) levels. In the multicenter randomized trial {"type":"clinical-trial","attrs":{"text":"NCT01562080","term_id":"NCT01562080"}}NCT01562080, there was large interindividual variability in the plasma LDLc response to AP supplementation. We hypothesized that the variability in LDLc response to AP supplementation may be linked to LDLR and PCSK9 polymorphisms.

Material and Methods

We sequenced the LDLR 3′ and 5′ untranslated regions (UTR) and the PCSK9 5′ UTR of 102 participants with moderate hypercholesterolemia in trial {"type":"clinical-trial","attrs":{"text":"NCT01562080","term_id":"NCT01562080"}}NCT01562080. In this trial, 50 individuals were treated with AP supplementation and the rest with placebo.

Results

Multiple linear regression analysis, using the response of LDLc levels to AP as the dependent variable, revealed that polymorphisms rs2149041 (c.-3383C>G) in the PCSK9 5′ UTR and rs14158 (c.*52G>A) in the LDLR 3′ UTR explained 14.1% and 6.4%, respectively, of the variability after adjusting for gender, age, and BMI of individuals. Combining polymorphisms rs2149041 and rs14158 explained 20.5% of this variability (p < 0.004).

Conclusions

Three polymorphisms in the 3′ UTR region of LDLR, c.*52G>A, c.*504G>A, and c.*773A>G, and two at the 5′ UTR region of PCSK9, c.−3383C>G and c.−2063A>G, were associated with response to AP. These results could explain the variability observed in the response to berberine among people with moderate hypercholesterolemia, and they may be useful in identifying patients who could potentially benefit from supplementation with AP.     

A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60–65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants.MethodsUsing short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients.ResultsAll PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method.ConclusionsWe developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted.     

Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.     

Encholirium pulchrum and E. diamantinum spp. nov. (Bromeliaceae) from the Espinhaço Range,Minas Gerais,Brazil

Two new species of Encholirium from the Espinhaço range, restricted to the Diamantina plateau of Minas Gerais state, Encholirium pulchrum Forzza, Leme & O. B. C. Ribeiro and Encholirium diamantinum Forzza, are described and illustrated, and their morphological characters are discussed and compared to those of close relatives.     

Microlicia crassa and M. maculata spp. nov. (Melastomataceae) from Minas Gerais,Brazil: morphology and leaf anatomy

Two new species of Microlicia from campos rupestres of Minas Gerais are described, illustrated and compared with their relatives. Microlicia crassa sp. nov. is similar to M. formosa, and M. maculata sp. nov. is similar to M. isophylla and M. tetrasticha. The trichomes covering both surfaces of the leaves of the two new species and their relative, M. isophylla may be characterized as sessile glands under a stereomicroscope, but anatomical studies revealed the presence of a short stalk. Microlicia crassa and M. maculata are assessed as “Endangered”, according to the IUCN categories and criteria.     

A new species of <Emphasis Type="Italic">Orthophytum</Emphasis> (Bromeliaceae,Bromelioideae) from Minas Gerais,Brazil

Orthophytum vidaliorum, a new species from Minas Gerais, Brazil, is described and illustrated, and its relationship to Orthophytum itambense is discussed.     

A new species of Passiflora (Passifloraceae) from Minas Gerais, Brazil

Passiflora boticarioana, a new species of subgen.Passiflora, supersect.Stipulata, sect.Dysosmia, from the state of Minas Gerais, Brazil, is described, illustrated, and compared with morphologically similar species.     

Molecular characterization of Polish patients with familial hypercholesterolemia: novel and recurrentLDLR mutations

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis ofLDLR andAPOB was performed in a group of 378 unrelated ADH patients, to explore the mutation spectrum that causes hypercholesterolemia in Poland. All patients were clinically diagnosed with ADH according to a uniform protocol and internationally accepted WHO criteria. Mutational analysis included all exons, exon-intron boundaries and the promoter sequence of theLDLR, and a fragment of exon 26 ofAPOB. Additionally, the MLPA technique was applied to detect rearrangements withinLDLR. In total, 100 sequence variations were identified in 234 (62%) patients. WithinLDLR, 40 novel and 59 previously described sequence variations were detected. Of the 99LDLR sequence variations, 71 may be pathogenic mutations. The most frequentLDLR alteration was a point mutation p.G592E detected in 38 (10%) patients, followed by duplication of exons 4–8 found in 16 individuals (4.2%). Twenty-five cases (6.6%) demonstrated the p.R3527Q mutation ofAPOB. Our findings imply that major rearrangements of theLDLR gene as well as 2 point mutations (p.G592E inLDLR and p.R3527Q inAPOB) are frequent causes of ADH in Poland. However, the heterogeneity ofLDLR mutations detected in the studied group confirms the requirement for complex molecular studies of Polish ADH patients.     

Epipolymorphisms within lipoprotein genes contribute independently to plasma lipid levels in familial hypercholesterolemia

Gene polymorphisms associated so far with plasma lipid concentrations explain only a fraction of their heritability, which can reach up to 60%. Recent studies suggest that epigenetic modifications (DNA methylation) could contribute to explain part of this missing heritability. We therefore assessed whether the DNA methylation of key lipoprotein metabolism genes is associated with high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride levels in patients with familial hypercholesterolemia (FH). Untreated FH patients (61 men and 37 women) were recruited for the measurement of blood DNA methylation levels at the ABCG1, LIPC, PLTP and SCARB1 gene loci using bisulfite pyrosequencing. ABCG1, LIPC and PLTP DNA methylation was significantly associated with HDL-C, LDL-C and triglyceride levels in a sex-specific manner (all P < 0.05). FH subjects with previous history of coronary artery disease (CAD) had higher LIPC DNA methylation levels compared with FH subjects without CAD (P = 0.02). Sex-specific multivariable linear regression models showed that new and previously reported epipolymorphisms (ABCG1-CpGC3, LIPC-CpGA2, mean PLTP-CpGC, LPL-CpGA3, CETP-CpGA2, and CETP-CpGB2) significantly contribute to variations in plasma lipid levels (all P < 0.001 in men and P < 0.02 in women), independently of traditional predictors such as age, waist circumference, blood pressure, fasting plasma lipids and glucose levels. These results suggest that epigenetic perturbations of key lipoprotein metabolism genes are associated with plasma lipid levels, contribute to the interindividual variability and might partially explain the missing heritability of plasma lipid levels, at least in FH.     

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.     

Using genetic variants to evaluate the causal effect of cholesterol lowering on head and neck cancer risk: A Mendelian randomization study

Head and neck squamous cell carcinoma (HNSCC), which includes cancers of the oral cavity and oropharynx, is a cause of substantial global morbidity and mortality. Strategies to reduce disease burden include discovery of novel therapies and repurposing of existing drugs. Statins are commonly prescribed for lowering circulating cholesterol by inhibiting HMG-CoA reductase (HMGCR). Results from some observational studies suggest that statin use may reduce HNSCC risk. We appraised the relationship of genetically-proxied cholesterol-lowering drug targets and other circulating lipid traits with oral (OC) and oropharyngeal (OPC) cancer risk using two-sample Mendelian randomization (MR). For the primary analysis, germline genetic variants in HMGCR, NPC1L1, CETP, PCSK9 and LDLR were used to proxy the effect of low-density lipoprotein cholesterol (LDL-C) lowering therapies. In secondary analyses, variants were used to proxy circulating levels of other lipid traits in a genome-wide association study (GWAS) meta-analysis of 188,578 individuals. Both primary and secondary analyses aimed to estimate the downstream causal effect of cholesterol lowering therapies on OC and OPC risk. The second sample for MR was taken from a GWAS of 6,034 OC and OPC cases and 6,585 controls (GAME-ON). Analyses were replicated in UK Biobank, using 839 OC and OPC cases and 372,016 controls and the results of the GAME-ON and UK Biobank analyses combined in a fixed-effects meta-analysis. We found limited evidence of a causal effect of genetically-proxied LDL-C lowering using HMGCR, NPC1L1, CETP or other circulating lipid traits on either OC or OPC risk. Genetically-proxied PCSK9 inhibition equivalent to a 1 mmol/L (38.7 mg/dL) reduction in LDL-C was associated with an increased risk of OC and OPC combined (OR 1.8 95%CI 1.2, 2.8, p = 9.31 x10^-05), with good concordance between GAME-ON and UK Biobank (I² = 22%). Effects for PCSK9 appeared stronger in relation to OPC (OR 2.6 95%CI 1.4, 4.9) than OC (OR 1.4 95%CI 0.8, 2.4). LDLR variants, resulting in genetically-proxied reduction in LDL-C equivalent to a 1 mmol/L (38.7 mg/dL), reduced the risk of OC and OPC combined (OR 0.7, 95%CI 0.5, 1.0, p = 0.006). A series of pleiotropy-robust and outlier detection methods showed that pleiotropy did not bias our findings. We found limited evidence for a role of cholesterol-lowering in OC and OPC risk, suggesting previous observational results may have been confounded. There was some evidence that genetically-proxied inhibition of PCSK9 increased risk, while lipid-lowering variants in LDLR, reduced risk of combined OC and OPC. This result suggests that the mechanisms of action of PCSK9 on OC and OPC risk may be independent of its cholesterol lowering effects; however, this was not supported uniformly across all sensitivity analyses and further replication of this finding is required.     

Four new species of <Emphasis Type="Italic">Chionolaena</Emphasis> (Asteraceae: Gnaphalieae) from south-eastern Brazil

Four new species of Chionolaena (C. adpressifolia, C. campestris, C. canastrensis and C. juniperina) are described and illustrated from Minas Gerais and S?o Paulo States, Brazil and their affinities assessed. A key to Chionolaena in South America is given.     

Three new species of <Emphasis Type="Italic">Eupatorieae</Emphasis> (Compositae) from Brazil

Three new species of Eupatorieae (Asteraceae) were collected in the “campos rupestres” of the Cadeia do Espinha?o, Minas Gerais state, Brazil, in the Itacolomi State Park, located between the cities of Ouro Preto and Mariana, Minas Gerais, Brazil, during a floristic survey focusing on Compositae in this area. Eupatorium semiamplexifolium, Mikania badiniana, and Stevia alexii are described and illustrated.     

Angiosperm epiphytes as conservation indicators in forest fragments: A case study from southeastern Minas Gerais, Brazil

Epiphytes play an important role in the flora and ecology of the tropical regions. Most floristic studies within the state of Minas Gerais focus on terrestrial, woody plant diversity, but this is a different approach, looking at epiphytic angiosperms in three highland areas in Southeastern Minas Gerais, namely Mata do Baú (MB), Reserva Biológica da Represa do Grama (RBRG) and Parque Estadual de Ibitipoca (PEI). Regular collections were performed in these sites between 1999 and 2007, complemented by herbarium records for PEI, and 181 species of epiphytic angiosperms were recorded in 66 genera, within 12 families. Orchidaceae, with 89 species, was by far the largest, and the most species rich genera were Peperomia (Piperaceae) and Pleurothallis s.l. (Orchidaceae), with 12 species each. Similarity analysis has shown a closer relationship between MB and RBRG, both composed by seasonal semideciduous forest, however, Jaccard (0.163) index are low. A similarity analysis including other 21 areas of southeastern and southern Brazil revealed strong influence of sazonality, vegetation type and altitude in the composition of the epiphytic flora, and a relative independence regarding the geographic proximity of the areas sampled. From the conservation standpoint, 30 species recorded for this work appear in the red list for the state of Minas Gerais, under different conservation categories. The low similarity indices obtained between the studied areas underline the importance of the conservation of each one of the remaining forest fragments in Minas Gerais, as their relative geographical proximity does not necessarily mean that their epiphytic flora is similar.     

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.