OPRM1 genotype and naltrexone response in depressed alcohol-dependent patients.

A functional polymorphism rs1799971 (A118G) in the µ-opioid receptor gene (OPRM1) produces an amino acid substitution Asn40Asp, which is believed to influence naltrexone response in nondepressed alcohol-dependent patients. In this study, patients with alcohol dependence and major depression (n=108) received open-label naltrexone and clinical case management for 12 weeks, and were randomized to citalopram or placebo. General linear mixed models examined the effect of the OPRM1 A118G genotype on alcohol outcomes during treatment. There was no evidence of any difference in the percentage of days abstinent, drinks per drinking day or percentage of heavy drinking days between Asp40 carriers and noncarriers during treatment. This study therefore failed to replicate the previous positive findings for this single nucleotide polymorphism in relation to naltrexone response, possibly indicating that the effect is not present in depressed patients.

Multiplexed Nanopore Sequencing of HLA-B Locus in Maori and Pacific Island Samples.

The human leukocyte antigen (HLA) system encodes the human major histocompatibility complex (MHC). HLA-B is the most polymorphic gene in the MHC class I region and many HLA-B alleles have been associated with adverse drug reactions (ADRs) and disease susceptibility. The frequency of such HLA-B alleles varies by ethnicity, and therefore it is important to understand the prevalence of such alleles in different population groups. Research into HLA involvement in ADRs would be facilitated by improved methods for genotyping key HLA-B alleles. Here, we describe an approach to HLA-B typing using next generation sequencing (NGS) on the MinION™ nanopore sequencer, combined with data analysis with the SeqNext-HLA software package. The nanopore sequencer offers the advantages of long-read capability and single molecule reads, which can facilitate effective haplotyping. We developed this method using reference samples as well as individuals of New Zealand Maori or Pacific Island descent, because HLA-B diversity in these populations is not well understood. We demonstrate here that nanopore sequencing of barcoded, pooled, 943 bp polymerase chain reaction (PCR) amplicons of 49 DNA samples generated ample read depth for all samples. HLA-B alleles were assigned to all samples at high-resolution with very little ambiguity. Our method is a scaleable and efficient approach for genotyping HLA-B and potentially any other HLA locus. Finally, we report our findings on HLA-B genotypes of this cohort, which adds to our understanding of HLA-B allele frequencies among Maori and Pacific Island people.

Impact of annual targeted treatment on infectious trachoma and susceptibility to reinfection.

CONTEXT: The World Health Organization developed the SAFE strategy (Surgery for trichiasis; Antibiotics for Chlamydia trachomatis infection; Facial cleanliness; and Environmental improvement) to eliminate blinding trachoma globally by the year 2020. Despite a number of studies using various intervals of treatment for different prevalence rates, there has been a lack of sufficient follow-up beyond the final treatment point to determine rates of recurrence of disease and infection and the risk factors that may contribute to each. OBJECTIVE: To evaluate the impact of 2 annual targeted azithromycin treatments on active trachoma and C trachomatis infection rates over 3 years in Vietnam. DESIGN, SETTING, AND PARTICIPANTS: Three communes were randomly selected for a longitudinal study in Vietnam from November 2000 through November 2003. Individuals (n = 3186) were graded for trachoma followed by conjunctival sampling to detect chlamydiae by commercial polymerase chain reaction. Grading and chlamydial detection were repeated every 6 months for 3 years. INTERVENTION: Azithromycin was given to children aged 5 through 15 years with active trachoma and their household members in SAFE and SA communes at baseline and 12 months; these communes were compared with the S-only control commune that did not receive azithromycin targeted treatment. MAIN OUTCOME MEASURES: Prevalence and incidence of active trachoma and C trachomatis infection in all communes at baseline, 6, 12, 18, 24, and 36 months. Subgroup analysis evaluated new infection, continuing infection, and reinfection at 6, 12, 18, 24, and 36 months and risk factors for each. RESULTS: Reinfection rates increased significantly between 12 and 36 months for SAFE (from 1.6 to 29.3 per 1000; P<.001) and SA (5.1 to 25.3 per 1000; P = .002) communes but not for the S-only commune (13.4 to 6.7 per 1000; P = .55) after 24 months. Compared with the S-only commune, mixed-effects and generalized estimating equations (GEE) logistic models showed that reinfection risk was significantly higher for SAFE (odds ratio [OR], 4.1; 95% confidence interval [CI], 1.5-9.8; P = .005) and SA (OR, 4.2; 95% CI, 1.1-17.3; P = .04) communes at 36 months. CONCLUSIONS: Increasing reinfection rates suggest that treatment may interrupt the duration of infection required for developing immunity, increasing the number of individuals susceptible to reinfection and adversely affecting disease prevalence over time. Additional research is needed to determine optimal trachoma control strategies, including evaluation of the "F" and "E" components. TRIAL REGISTRATION: www.actr.org.au Identifier: 12606000360516.

Cross-Comparison of Exome Analysis, Next-Generation Sequencing of Amplicons, and the iPLEX(®) ADME PGx Panel for Pharmacogenomic Profiling.

Whole-exome sequencing (WES) has been widely used for analysis of human genetic diseases, but its value for the pharmacogenomic profiling of individuals is not well studied. Initially, we performed an in-depth evaluation of the accuracy of WES variant calling in the pharmacogenes CYP2D6 and CYP2C19 by comparison with MiSeq(®) amplicon sequencing data (n = 36). This analysis revealed that the concordance rate between WES and MiSeq(®) was high, achieving 99.60% for variants that were called without exceeding the truth-sensitivity threshold (99%), defined during variant quality score recalibration (VQSR). Beyond this threshold, the proportion of discordant calls increased markedly. Subsequently, we expanded our findings beyond CYP2D6 and CYP2C19 to include more genes genotyped by the iPLEX(®) ADME PGx Panel in the subset of twelve samples. WES performed well, agreeing with the genotyping panel in approximately 99% of the selected pass-filter variant calls. Overall, our results have demonstrated WES to be a promising approach for pharmacogenomic profiling, with an estimated error rate of lower than 1%. Quality filters, particularly VQSR, are important for reducing the number of false variants. Future studies may benefit from examining the role of WES in the clinical setting for guiding drug therapy.

Mutation analysis of the carbohydrate sulfotransferase gene in Vietnamese with macular corneal dystrophy.

PURPOSE: Mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal N-acetylglucosamine-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of macular corneal dystrophy (MCD) in various ethnicities. This study was conducted to examine the CHST6 gene in Vietnamese with MCD. METHODS: Nineteen unrelated families, including 35 patients and 38 unaffected relatives were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals served as control subjects. Genomic DNA was extracted from leukocytes. Analysis of the CHST6 gene was performed with polymerase chain reaction and direct sequencing. Corneal buttons were studied histopathologically. RESULTS: A slit lamp examination revealed clinical features of MCD with gray-white opacities and stromal haze between. On histopathology, corneal sections showed positive staining with colloidal iron. Sequencing of the CHST6 gene revealed six homozygous and three compound heterozygous mutations. The homozygous mutations, including L59P, V66L, R211Q, W232X, Y268C, and 1067-1068ins(GGCCGTG) were detected, respectively, in two, one, eight, one, one, and two families. Compound heterozygous mutations R211Q/Q82X, S51L/Y268C, and Y268C/1067-1068ins(GGCCGTG) were identified, each in one family. A single heterozygous change at codon 76 (GTG-->ATG) was detected in family L, resulting in a valine-to-methionine substitution (V76M). None of these mutations was detected in the control group. CONCLUSIONS: Mutations identified in the CHST6 gene cosegregated with the disease phenotype in all but one family studied and thus caused MCD. Among these, the R211Q detected in 9 of 19 families may be the most common mutation in Vietnamese. These data also indicate that significant allelic heterogeneity exists for MCD.

A novel mutation of M1S1 gene found in a Vietnamese patient with gelatinous droplike corneal dystrophy.

To identify the genetic defect in the M1S1 gene responsible for gelatinous droplike corneal dystrophy (GDLD) in a Vietnamese family.Experimental study. Blood samples were collected from a patient and the unaffected members of a GDLD-affected family. Fifty normal unrelated subjects of Vietnamese origin were used as controls. Genomic DNA was extracted from blood leukocytes. DNA analysis of the M1S1 gene was performed using polymerase chain reaction and direct sequencing. Sequencing of the M1S1 gene revealed a deletion of a 12-base-pair (bp) fragment from nucleotide positions 772 to 783 [772 to 783del(ATCTATTACCTG)], resulting in a loss of four amino acids at codons 258 to 261 (L258-liter261del). Yet, an insertion of nucleotide T in place of the missing sequence (772insT) was found. This combined mutation was homozygous in the GDLD-affected patient and heterozygous in his unaffected son and younger sister. Such genetic alteration was excluded in the control population. This is the first report of a mutational analysis performed in a Vietnamese patient with GDLD. In this family, the novel 772 to 783del(ATCTATTACCTG) + 772insT mutation on the M1S1 gene was well cosegregated with the phenotype and thus expected to cause GDLD. Although the M1S1 gene was responsible for GDLD in Vietnamese patients, the mutation found here is completely different from that previously reported in Japanese patients, where GDLD is most frequently seen.

Identification of novel mutations of the CHST6 gene in Vietnamese families affected with macular corneal dystrophy in two generations.

PURPOSE: To report the clinical and genetic findings of Vietnamese families affected with macular corneal dystrophy (MCD) in 2 generations. METHODS: Two families, including 7 patients and 3 unaffected members, were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals were used as controls. Genomic DNA was extracted from leukocytes. Analysis of the carbohydrate sulfotransferase (CHST6) gene was performed using polymerase chain reaction and direct sequencing. RESULTS: The typical form of MCD was recognized in family B, in which sequencing of CHST6 gene revealed an nt 1067-1068ins(GGCCGTG) mutation (frameshift after 125V) homozygously in MCD patients and heterozygously in the unaffected members. Family N also showed clinical features of MCD, moderate in the mother but severe in the affected son. Sequencing revealed a single heterozygous Arg211Gln in the mother, compound heterozygous Arg211Gln+ Gln82Stop in the affected son, and heterozygous Arg211Gln mutation in the unaffected members. The identified mutations in these pedigrees were excluded from normal controls. CONCLUSIONS: The novel frameshift and compound heterozygous mutations might be responsible for MCD in the families studied. The phenotypic variation between affected parents and offspring was unclear. In family N, severe MCD phenotype seen in the affected son may be due the fact that he had an early stop codon mutation (Gln82Stop).

A novel mutation of the TGFBI gene found in a Vietnamese family with atypical granular corneal dystrophy.

BACKGROUND: Mutation of the human transforming growth factor beta-induced (TGFBI) gene causes granular corneal dystrophy (GCD) in various ethnic groups. In this report, we identify the genetic defect on the TGFBI gene in a Vietnamese family with atypical GCD . CASES: The patient and her relatives were examined clinically. Genomic DNA was extracted from blood leukocytes. Fifty normal Vietnamese were used as controls. Analysis of the TGFBI gene was performed using polymerase chain reaction and direct sequencing. OBSERVATIONS: The 42-year-old proband clinically showed multiple white dot-like opacities scattered in the anterior and mid-stroma of the central cornea. Unlike GCD, these deposits were smaller, localized deeper and less severe. DNA analysis revealed a nucleotide transversion at codon 123 (GAC --> CAC), causing Asp --> His substitution (D123H). This mutation was also detected in 3 out of 5 unaffected family members, but was absent in the 50 normal controls. CONCLUSIONS: The novel D123H mutation of the TGFBI gene was not co-segregated with GCD in the family studied, and did not exist in the control population. It probably was a disease-causing mutation, thus expected to cause a novel variant of GCD in the proband. The detection of the D123H mutation in three unaffected family members indicates that it has low penetrance for GCD.

Mutation analysis of the TGFBI gene in Vietnamese with granular and Avellino corneal dystrophy.

PURPOSE: Mutations of the human transforming growth factor beta-induced gene (TGFBI) were reported to cause granular (GCD) and Avellino (ACD) corneal dystrophy in various nationalities. In this study we examined the TGFBI gene in a Vietnamese population with GCD and ACD. METHODS: Eight unrelated Vietnamese families, including 20 affected and 24 unaffected individuals, were examined; 50 normal Vietnamese individuals were used as controls. Genomic DNA was extracted from peripheral blood leukocytes. The TGFBI gene was analyzed using the polymerase chain reaction and direct sequencing. The corneal button was studied. RESULTS: Slit-lamp examination revealed typical features of GCD in most cases. A few features of ACD and a patient with an atypical form of GCD were also seen. Histopathological analysis of a GCD cornea showed deposits that stained bright red with Masson trichrome. Sequencing revealed three distinct mutations: R555W in six families, R124H in one family, and D123H in another. CONCLUSIONS: R555W and R124H mutations were co-segregated with the disease phenotype and thus caused GCD and ACD, respectively, in the families studied. The R555W detected in six of the eight families indicates that the GCD phenotype may be the most common in Vietnamese individuals, unlike in other Asians (Japanese and Korean), where ACD is most common (>90%). The D123H mutation may cause an atypical variant of GCD.