Deletion of CTCF sites in the SHH locus alters enhancer-promoter interactions and leads to acheiropodia.

Acheiropodia, congenital limb truncation, is associated with homozygous deletions in the LMBR1 gene around ZRS, an enhancer regulating SHH during limb development. How these deletions lead to this phenotype is unknown. Using whole-genome sequencing, we fine-mapped the acheiropodia-associated region to 12 kb and show that it does not function as an enhancer. CTCF and RAD21 ChIP-seq together with 4C-seq and DNA FISH identify three CTCF sites within the acheiropodia-deleted region that mediate the interaction between the ZRS and the SHH promoter. This interaction is substituted with other CTCF sites centromeric to the ZRS in the disease state. Mouse knockouts of the orthologous 12 kb sequence have no apparent abnormalities, showcasing the challenges in modelling CTCF alterations in animal models due to inherent motif differences between species. Our results show that alterations in CTCF motifs can lead to a Mendelian condition due to altered enhancer-promoter interactions.

Human Induced Pluripotent Stem Cell Derived Sensory Neurons are Sensitive to the Neurotoxic Effects of Paclitaxel.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell-derived sensory neuron (iPSC-SN) model was developed for the study of chemotherapy-induced neurotoxicity. The iPSC-SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca2+ influx in response to capsaicin, α,ß-meATP, and glutamate. The iPSC-SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half-maximal inhibitory concentration (IC50 ) values of 38.1 µM (95% confidence interval (CI) 22.9-70.9 µM) for 48-hour exposure and 9.3 µM (95% CI 5.7-16.5 µM) for 72-hour treatment. Paclitaxel causes dose-dependent and time-dependent changes in neurite network complexity detected by ßIII-tubulin staining and high content imaging. The IC50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3-16.9 µM) for 48-hour exposure and 0.6 µM (95% CI 0.09-9.9 µM) for 72-hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate-induced neuronal excitability were also observed with paclitaxel exposure. The iPSC-SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC-SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy-induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment.

Genomewide Meta-Analysis Validates a Role for S1PR1 in Microtubule Targeting Agent-Induced Sensory Peripheral Neuropathy.

Microtubule targeting agents (MTAs) are anticancer therapies commonly prescribed for breast cancer and other solid tumors. Sensory peripheral neuropathy (PN) is the major dose-limiting toxicity for MTAs and can limit clinical efficacy. The current pharmacogenomic study aimed to identify genetic variations that explain patient susceptibility and drive mechanisms underlying development of MTA-induced PN. A meta-analysis of genomewide association studies (GWAS) from two clinical cohorts treated with MTAs (Cancer and Leukemia Group B (CALGB) 40502 and CALGB 40101) was conducted using a Cox regression model with cumulative dose to first instance of grade 2 or higher PN. Summary statistics from a GWAS of European subjects (n = 469) in CALGB 40502 that estimated cause-specific risk of PN were meta-analyzed with those from a previously published GWAS of European ancestry (n = 855) from CALGB 40101 that estimated the risk of PN. Novel single nucleotide polymorphisms in an enhancer region downstream of sphingosine-1-phosphate receptor 1 (S1PR1 encoding S1PR1 ; e.g., rs74497159, ßCALGB 40101 per allele log hazard ratio (95% confidence interval (CI)) = 0.591 (0.254-0.928), ßCALGB 40502 per allele log hazard ratio (95% CI) = 0.693 (0.334-1.053); PMETA  = 3.62 × 10-7 ) were the most highly ranked associations based on P values with risk of developing grade 2 and higher PN. In silico functional analysis identified multiple regulatory elements and potential enhancer activity for S1PR1 within this genomic region. Inhibition of S1PR1 function in induced pluripotent stem cell-derived human sensory neurons shows partial protection against paclitaxel-induced neurite damage. These pharmacogenetic findings further support ongoing clinical evaluations to target S1PR1 as a therapeutic strategy for prevention and/or treatment of MTA-induced neuropathy.

P-Glycoprotein Inhibition Exacerbates Paclitaxel Neurotoxicity in Neurons and Patients With Cancer.

Paclitaxel-induced peripheral neuropathy (PIPN) is a common and dose-limiting adverse event. The role of P-glycoprotein (P-gp) in the neuronal efflux of paclitaxel was assessed using a translational approach. SH-SY5Y cells were differentiated to neurons and paclitaxel toxicity in the absence and presence of a P-gp inhibitor was determined. Paclitaxel caused marked dose-dependent toxicity in SH-SY5Y-derived neurons. Paclitaxel neurotoxicity was exacerbated with concomitant P-gp inhibition by valspodar and verapamil, consistent with increased intracellular accumulation of paclitaxel. Patients with cancer treated with paclitaxel and P-gp inhibitors had a 2.4-fold (95% confidence interval (CI) 1.3-4.3) increased risk of peripheral neuropathy-induced dose modification while a 4.7-fold (95% CI 1.9-11.9) increased risk for patients treated with strong P-gp inhibitors was observed, and a 7.0-fold (95% CI 2.3-21.5) increased risk in patients treated with atorvastatin. Atorvastatin also increased neurotoxicity by paclitaxel in SH-SY5Y-derived neurons. Clinicians should be aware that comedication with P-gp inhibitors may lead to increased risk of PIPN.

Saturation mutagenesis of twenty disease-associated regulatory elements at single base-pair resolution.

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

Integration of multiple epigenomic marks improves prediction of variant impact in saturation mutagenesis reporter assay.

The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.

Polymorphism in ERCC1 confers susceptibility of coronary artery disease and severity of coronary artery atherosclerosis in a Chinese Han population.

Excision repair cross-complementing 1 (ERCC1) gene encodes ERCC1 protein, which is mainly responsible for the repair of DNA damage in different diseases including coronary artery atherosclerosis by acting as a rate-limiting element in nucleotide excision repair (NER). Using a three-stage case-control study with 3037 coronary artery disease (CAD) patients and 3002 controls, we investigated associations of three single nucleotide polymorphisms (SNPs) with CAD risk and severity of coronary artery atherosclerosis in Chinese Han population. In the discovery set, the variant allele T of rs11615 was significantly associated with higher CAD risk (adjusted OR = 1.27, P = 0.006) and severity of coronary artery atherosclerosis (adjusted OR = 1.54, P = 0.003). These associations were more remarkable in the merged set (adjusted OR = 1.23, P = 8 × 10-6 for CAD risk; adjusted OR = 1.36, P = 4.3 × 10-5 for severity of coronary artery atherosclerosis). And the expression level of ERCC1 was significantly higher in CAD cases than controls. Multiplicative interactions among SNP rs11615, alcohol drinking, history of T2DM, and history of hyperlipidemia could increase 5.06-fold risk of CAD (P = 1.59 × 10-9). No significant association of rs2298881 and rs3212986 with CAD risk was identified. Taken together, SNP rs11615 in ERCC1 gene might confer susceptibility to CAD and severity of coronary atherosclerosis in a Chinese Han population.

miR-22 targets YWHAZ to inhibit metastasis of hepatocellular carcinoma and its down-regulation predicts a poor survival.

Many miRNAs are associated with the carcinogenesis of hepatocellular carcinoma (HCC) and some exhibit potential prognostic value. In this study, to further confirm the prognostic value of miRNAs in HCC, we employed miRNA-sequencing data of tumor tissues of 372 HCC patients released by The Cancer Genome Atlas (TCGA) and identified 3 miRNAs including miR-22, miR-9-1 and miR-9-2 could be used as independent predictors for HCC prognostic evaluation. As a tumor-suppressive miRNA, miR-22 was down-regulated in HCC tissues. This down-regulation correlated with tumor vascular invasion, Edmondson-Steiner grade, TNM stage, and AFP level. Moreover, biofunctional investigations revealed that miR-22 significantly attenuated cellular proliferation, migration and invasion of HCC cells. Additionally, through gene expression profiles and bioinformatics analysis, YWHAZ was identified to be a direct target of miR-22 and its overexpression partially counteracted the inhibitory effects of miR-22 on HCC cells. Finally, molecular studies further confirmed that miR-22 promoted the accumulation of FOXO3a in nucleus and subsequently reversed invasive phenotype of HCC cells by repressing YWHAZ-mediated AKT phosphorylation. Taken together, these data demonstrate that miR-22 exhibits tumor-suppressive effects in HCC cells by regulating YWHAZ/AKT/FOXO3a signaling and might be used as an independent prognostic indicator for HCC patients.

Deletion of Pr130 Interrupts Cardiac Development in Zebrafish.

Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction (EC) coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening). Hematoxylin and eosin (H&E) staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT). Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

Risk-Association of DNMT1 Gene Polymorphisms with Coronary Artery Disease in Chinese Han Population.

Recently, a significant epigenetic component in the pathogenesis of Coronary Artery Disease (CAD) has been realized. Here, we evaluated the possible association of candidate Single Nucleotide Polymorphisms (SNPs) in the epigenetic-regulatory gene, DNA methyltransferase 1 (DNMT1), with CAD in Chinese Han population. Five tag SNPs (rs16999593, rs2336691, rs2228611, rs4804494, rs7253062) were analyzed by High Resolution Melt (HRM) method in 476 CAD patients and 478 controls. Overall, there were significant differences in the genotype and allele distributions of rs2228611 and rs2336691, between patients and controls. The minor A allele of rs2228611 was associated with a lower risk of CAD (p = 0.034); modest effect in the additive analysis but also marginal significance was found in the recessive model [ORadditive = 0.404 (0.184, 0.884), p = 0.023 and ORrecessive = 0.452 (0.213, 0.963), p = 0.040] after adjusting for confounders. While the rs2336691 A allele were associated with a higher risk of developing CAD (p = 0.037); borderline significant association in both additive and dominant models [ORadditive = 1.632 (1.030, 2.583), p = 0.037 and ORdominant = 1.599 (1.020, 2.507), p = 0.040]. In conclusion, these data provide the first evidence that occurrence of CAD may be moderated by genetic variation in the gene involved in the epigenetic machinery.

Common rs7138803 variant of FAIM2 and obesity in Han Chinese.

BACKGROUND: Obesity causes severe healthcare problem worldwide leading to numerous diseases, such as cardiovascular diseases and diabetes mellitus. Previous Genome-Wide Association Study (GWAS) identified an association between a single nucleotide polymorphism (SNP) rs7138803, on chromosome 12q13 and obesity in European Caucasians. Since the genetic architecture governing the obesity may vary among different populations, we investigate the variant rs7138803 in Chinese population to find out whether it is associated with obesity. METHODS: A population-based cohort association study was carried out using the High Resolution Melt (HRM) method with 1851 participants. The association between rs7138803 genotypes and body mass index (BMI) was modeled with a general linear model, and a case-control study for the association between rs7138803 genotypes and obesity was performed using Pearson's χ2 test. There was no indication of a deviation from Hardy-Weinberg equilibrium (HWE p value = 0.51) in our sample. RESULTS: No association was detected between SNP rs7138803 and BMI in our Chinese Han population with a P value of 0.51. SNP rs7138803 was found to be not associated with common forms of obesity after adjusting for age and sex in the Chinese population. SNP rs7138803 was not associated with other obesity related traits, including T2DM, hypertension, lipid profiles, and ischemic stroke. CONCLUSION: Our data suggest that the rs7138803 exerts no significant effect on obesity in Chinese Han population. Larger cohorts may be more appropriate to detect an effect of this SNP on common obesity.

Dual-responsive gold nanoparticles for colorimetric recognition and testing of carbohydrates with a dispersion-dominated chromogenic process.

A dispersion-dominated colorimetric approach for the recognition of carbohydrates based on biomolecule-responsive AuNPs is presented. Taking advantage of the unique dual-responsiveness of smart copolymers, the aggregation and dispersion of AuNPs can be modulated by both temperature and different kinds of carbohydrates, giving rise to a novel chromogenic mechanism for the recognition and testing of carbohydrates in aqueous media.

Investigation of the relationship between apolipoprotein E gene polymorphisms and hepatitis B virus infection in northern China.

BACKGROUND: Certain genetic polymorphisms can lead to differences in immunity function, resulting in different clinical outcomes for hepatitis B virus (HBV) patients. The aim of this study was to investigate the association between apolipoprotein E (ApoE) gene polymorphisms and HBV infection status in northern Chinese individuals. METHODS: Genomic DNA was extracted using an improved sodium iodide (NaI) method from the peripheral blood of 270 patients with hepatitis B and 112 healthy controls. Multiplex Amplification Refractory Mutation System (Multi-ARMS) was performed to analyze ApoE gene polymorphisms with three alleles (ɛ2, ɛ3, ɛ4) in patients and controls. A chemiluminescence assay was used to detect serological markers for hepatitis B infection status. RESULTS: An improved PCR system for the detection of ApoE gene polymorphisms was established successfully. The frequency of the ɛ2 allele in patients with HBV infection was higher than that of normal controls (p<0.05). The ɛ2 allele, compared with the ɛ3 and ɛ4 alleles, showed positive correlation with the different HBV infection models [odds ratio (OR)=1.735, 95% confidence interval (CI): 1.509-1.999, p<0.01; OR=1.768, 95% CI: 1.554-2.011, p<0.01]. The OR for the ApoE ɛ2 allele was 1.503 in a multivariate unconditional logistic regression model (OR=1.503, 95% CI: 1.212-1.754, p<0.01). CONCLUSIONS: Our results indicated that the ApoE gene polymorphism was associated with HBV infection, and the ɛ2 allele showed positive correlation with HBV infection in northern China.

The lectin-like domain of tumor necrosis factor improves lung function after rat lung transplantation--potential role for a reduction in reactive oxygen species generation.

OBJECTIVE: To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required. DESIGN: Prospective, randomized laboratory investigation. SETTING: University-affiliated laboratory. SUBJECTS: Adult female rats. INTERVENTIONS: Tuberoinfundibular peptide, mimicking the lectin-like domain of tumor necrosis factor, mutant TIP peptide, N,N'-diacetylchitobiose/TIP peptide, and amiloride/TIP peptide were instilled intratracheally in the left lung immediately before the isotransplantation was performed. An additional group received an intravenous TIP peptide treatment, 1.5 mins before transplantation. Studies using isolated rat type II alveolar epithelial cell monolayers and ovine pulmonary endothelial cells were also performed. MEASUREMENTS AND MAIN RESULTS: Intratracheal pretreatment of the transplantable left lung with the TIP peptide, but not with an inactive mutant TIP peptide, resulted in significantly improved oxygenation 24 hrs after transplantation. This treatment led to a significantly reduced neutrophil content in the lavage fluid. Both the effects on oxygenation and neutrophil infiltration were inhibited by the epithelial sodium channel blocker amiloride. The TIP peptide blunted reactive oxygen species production in pulmonary artery endothelial cells under hypoxia and reoxygenation and reduced reactive oxygen species content in the transplanted rat lungs in vivo. Ussing chamber experiments using monolayers of primary type II rat pneumocytes indicated that the primary site of action of the peptide was on the apical side of these cells. CONCLUSIONS: These data demonstrate that the TIP peptide significantly improves lung function after lung transplantation in the rat, in part, by reducing neutrophil content and reactive oxygen species generation. These studies suggest that the TIP peptide is a potential therapeutic agent against the ischemia reperfusion injury associated with lung transplantation.

The lectin-like domain of TNF protects from listeriolysin-induced hyperpermeability in human pulmonary microvascular endothelial cells - a crucial role for protein kinase C-alpha inhibition.

Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) alpha/beta inhibitor GO6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-alpha activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.

[Analysis on gene mutations in a Chinese pedigree with autosomal dominant inheritance cataract].

OBJECTIVE: The aim of this study was to reveal the genetic defect of the autosomal dominant inheritance cataract in a Chinese pedigree. METHODS: Case-control study. There were 26 individuals investigated with clinical examination in a Chinese four generations pedigree. The genome DNA of the individuals was extracted by the improved NaI method. The exons of six cataract candidate genes in 204 normal controls and 42 senile cataract patients were screened for the mutation by PCR restriction fragment length polymorphism (PCR-RFLP) methods. RESULTS: The phenotype of the cataract was pulverulent nuclear cataract. A novel C/T transition at nucleotide position 827 was identified in the GJA8 gene that led to a serine to phenylalanine change in codon 276. This mutation was not found in 42 senile cataract patients and in 204 controls. Four single nucleotide polymorphisms (SNPs) were also found in a cataract candidate gene in the family members. CONCLUSIONS: A novel GJA8 gene mutation was found in a Chinese autosomal dominant inheritance cataract pedigree. A substitution, C276T in GJA8 gene, was identified as the most likely causative mutation underlying the phenotype of pulverulent nuclear cataract in all affected family members.

Two mutations in LDLR gene were found in two Chinese families with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal dominantly inherited disease mainly caused by mutations of the gene encoding the low density lipoprotein receptor (LDLR) and Apolipoprotein (Apo) B. First the common mutation R3500Q in ApoB gene was determined using PCR/RFLP method. Then the LDLR gene was screened for mutations using Touch-down PCR, SSCP and sequencing techniques. Furthermore, the secondary structure of the LDLR protein was predicted with ANTHEPROT5.0. The R3500Q mutation was absent in these two families. A heterozygous p.W483X mutation of LDLR gene was identified in family A which caused a premature stop codon, while a homozygous mutation p.A627T was found in family B. The predicted secondary structures of the mutant LDLR were altered. We identified two known mutations (p.W483X, p.A627T) of the LDLR gene in two Chinese FH families respectively.

A novel connexin 50 (GJA8) mutation in a Chinese family with a dominant congenital pulverulent nuclear cataract.

PURPOSE: To identify the genetic cause responsible for the autosomal dominant hereditary cataract in a Chinese family. METHODS: A whole family of a proband who has a dominant congenital pulverulent nuclear cataract was recruited into Zhongnan Hospital. The lenses of patients were observed by a slit-lamp microscope, and the lenses of the proband's mother were analyzed by scanning electron microscopy. Mutation screening was performed in the cataract candidate genes coding for crystallins and connexin 50 by sequencing of polymerase chain reaction (PCR) products amplified from blood leukocyte DNA samples of eight family members. The identified mutation was then investigated in other participated family members, 200 normal controls, and 40 senile cataract patients by the restriction fragment length polymorphism (RFLP) method. RESULTS: The structure of the lens opacities of the proband's mother is puffy, and the fibers are tangled under a scanning electron microscope. A novel C>T transition at nucleotide position 827 was determined in the connexin 50 (GJA8) gene. This mutation led to a serine (S) to phenylalanine (F) amino acid substitution in amino acid position 276 where the secondary structure prediction suggested a helix replaced by a sheet. And the mutation was neither found in the 200 controls nor in the 40 senile cataract patients. CONCLUSIONS: A novel GJA8 gene mutation was found to be associated with hereditary cataract in a Chinese congenital cataract family.

[Mutation screening and functional analysis of low density lipoprotein receptor in a familial hypercholesterolemia family].

OBJECTIVE: To screen the mutations of the low density lipoprotein receptor (LDLR) gene in a familial hypercholesterolemia (FH) family, and analyze the LDL-uptaking function of LDLR on lymphocytes of patients. METHODS: Genomic DNA was extracted from four affected members in a Chinese FH family. The presence of apoB100 gene R3500Q mutation which results in familial defective apolipoprotein B100 (FDB) was excluded first. Fragments of the LDLR gene were amplified by touch-down polymerase chain reaction (Touch-down PCR) and analyzed by single-strand conformational polymorphism (SSCP). The suspect fragments of the LDLR gene were cloned and sequenced. Furthermore, the lymphocytes bounded with fluorescent-labeled LDL (DiI-LDL) were measured by fluorescence flow cytometry. RESULTS: A nonsense mutation was identified in exon 10 of LDLR gene. This mutation gave rise to a premature stop codon (W462X), resulting in the absence of most of the LDLR domains. It was detected in all the affected members of the FH family. The ratios of functional LDLR in lymphocytes from patients and normal controls were 63.7% and 77.3% respectively. As a result, the activity of the functional LDLR in patients was just 82.4% of that in the normal controls. CONCLUSION: It is possible that the W462X mutation of LDLR gene is the main cause for the disease in this family.

Study of Kir6.2/KCNJ11 gene in a sudden cardiac death pedigree.

In clinic, the patients with acute myocardial infarction (AMI) are at high risk to develop ischemia-induced ventricular arrhythmias leading to sudden cardiac death (SCD). Some studies suggest that individual susceptibility to ischemia-induced arrhythmia may be related to the genes encoding ion channels. One of them is the cardiac ATP-sensitive potassium channel (K(ATP)), which is an octamer composed of four pore-forming inwardly rectifying potassium-channel subunits (Kir6.2) and four regulatory sulfonylurea-receptor subunits (SUR2A). They play important roles in the physiology and pathophysiology of cardiovascular system by coupling the metabolic state of the cells to cellular electrical activity. So far, some mutations and polymorphisms of Kir6.2/KCNJ11 gene showed significant correlation with type 2 diabetes. But it was not sure whether it was associated with acute myocardial diseases. Hence a complete mutational analysis of Kir6.2/KCNJ11 gene was performed in a pedigree of sudden cardiac death. The complete coding region and the intron-exon boundaries of KCNJ11 were amplified from genomic DNA using polymerase chain reaction (PCR). Direct sequencing was done to identify any mutations and then further confirmed by restriction site polymorphism (RSP) approach. No mutation was detected in the samples analyzed, a common polymorphism K23E (A>G) was noticed in this pedigree and the proband showed a homozygote genotype (G/G). The result suggests that the Kir6.2/KCNJ11 gene is not related to sudden cardiac death in this family.