Molecular identification of HLA-B75 serotype markers by qPCR: A more inclusive pharmacogenetic approach before carbamazepine prescription.

Genetic screening for HLA-B*15:02 before prescribing carbamazepine is standard practice to prevent severe cutaneous adverse reactions in Asian populations. These reactions are associated not only with this allele but also with closely related HLA-B75 serotype markers-HLA-B*15:11 and HLA-B*15:21-which are prevalent in several Asian countries. However, a reliable method for identifying HLA-B75 serotype markers is still not available. We developed an in-house quantitative PCR (qPCR) for HLA-B75 screening and validated it using 303 anonymized DNA samples. Due to inadequate quality control, the qPCR results for 11 samples were excluded. We analyzed the sensitivity and specificity of the test using 93 HLA-typed samples. The concordance between the qPCR method and an established screening method was assessed using 199 HLA-screened samples tested for HLA-B*15:02 at Songklanagarind Hospital, Songkhla, Thailand. All discordant results were confirmed by Sanger sequencing. The qPCR method demonstrated a sensitivity of 100% (95% confidence interval = 83.16%-100.00%) and a specificity of 100% (95% confidence interval = 95.07%-100.00%). Concordance analysis revealed a 96.5% agreement between methods (192/199; 44 positive and 148 negative results). All discordant results were due to HLA-B75 markers not being HLA-B*15:02 (two samples with HLA-B*15:11 and five samples with HLA-B*15:21). In conclusion, this qPCR method could be useful for identifying HLA-B75 carriers at risk of carbamazepine-induced reactions in Asian populations where carriers of HLA-B*15:02, HLA-B*15:11, or HLA-B*15:21 are common.

Clinical and molecular characteristics of FMR1 microdeletion in patient with fragile X syndrome and review of the literature.

BACKGROUND: Fragile X syndrome (FXS) is mainly caused by FMR1 CGG repeat expansions. Other types of mutations, particularly deletions, are also responsible for FXS phenotypes, however these mutations are often missed by routine clinical testing. MATERIALS AND METHODS: Molecular diagnosis in cases of suspected FXS was a combination of PCR and Southern blot. Measurement of the FMRP protein level was useful for detecting potentially deleterious impact. RESULTS: PCR analysis and Southern blot revealed a case with premutation and suspected deletion alleles. Sanger sequencing showed that the deletion involved 313 bp upstream of repeats and some parts of CGG repeat tract, leaving transcription start site. FMRP was detected in 5.5 % of blood lymphocytes. CONCLUSION: According to our review of case reports, most patients carrying microdeletion and full mutation had typical features of FXS. To our knowledge, our case is the first to describe mosaicism of a premutation and microdeletion in the FMR1 gene. The patient was probably protected from the effects of the deletion by mosaicism with premutation allele, leading to milder phenotype. It is thus important to consider appropriate techniques for detecting FMR1 variants other than repeat expansions which cannot be detected by routine FXS diagnosis.

Significant associations between 5-hydroxytryptaminetransporter-linked promoter region polymorphisms of the serotonin transporter (solute carrier family 6 member 4) gene and Thai patients with autism spectrum disorder.

Autism spectrum disorder (ASD) is a form of pervasive developmental disorder manifested by impairment in social interactions and repetitive behaviors. Although genetic contribution is strongly suspected in autism, the specific genetic factors remain unidentified. Hyperserotoninemia has been reported in some autistic patients, and several studies have demonstrated an association between 5-hydroxytryptamine-transporter-linked promoter region (5-HTTLPR) polymorphisms and rs25531 single nucleotide polymorphism in the serotonin transporter gene (solute carrier family 6 member 4; SLC6A4) and ASD, indicating a possible involvement of the serotonin system in the etiology of ASD.To explore this situation further, a case-control association study of 5-HTTLPR and rs25531 polymorphisms on Thai ASD patients was conducted. A total of 188 ASD cases fulfilling the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria (156 males and 32 females) and a total of 250 normal controls were recruited from the same ethnic backgrounds. 5-HTTLPR polymorphisms (Long, L; Short, S) and rs25531 (A/G) single nucleotide polymorphism were genotyped and compared between the patients and normal controls using chi-square statistics.The L/L genotype was more common in patients than in the controls (13.8% vs 5.2%, P = .006), and the LA haplotype was found in patients more than the controls (16.9% vs 12.2%, P = .048). When male patients were analyzed alone (156 individuals), the associations were also statistically significant with P = .017 for L/L genotype, and P = .019 for LA haplotype distribution.Our findings support previous reports suggesting an association between the 5-HTTLPR and rs25531 polymorphisms of SLC6A4 and patients with ASD.

Mutation screening of the neurexin 1 gene in thai patients with intellectual disability and autism spectrum disorder.

AIM: Neurexin 1 has two major protein isoforms using alternative promoters, coding for the alpha-neurexin 1 (α-NRXN1) and beta-neurexin 1 (ß-NRXN1) genes. This study is to explore the possibility that variants of the NRXN1 gene predispose to intellectual disability (ID) and autism spectrum disorder (ASD). METHODS: The coding regions in 24 exons and exon-intron boundaries of the NRXN1 gene were investigated in 115 Thai patients with ID and ASD by direct DNA sequencing. RESULTS: Nine novel variants of the NRXN1 gene were identified. Four novel variants were found in the ß-NRXN1 gene, one variant of six GGC repeats in exon 1, and three variants at the 5'UTR. Five novel variants were identified in the α-NRXN1 gene, four intronic variants and one missense variant in exon 14 (c.2713T>A or p.F905I). CONCLUSION: Mutation screening of the NRXN1gene in patients with ID and ASD may be useful to identify potential variants predisposing to ID and ASD. However, further studies utilizing protein functional analysis of the novel variants are required for a more definite conclusion.

Mutation screening of the CDKL5 gene in cryptogenic infantile intractable epilepsy and review of clinical sensitivity.

PURPOSES: To perform CDKL5 mutation screening in Thai children with cryptogenic infantile intractable epilepsy and to determine the clinical sensitivity of CDKL5 screening when different inclusion criteria were applied. METHODS: Children with cryptogenic infantile intractable epilepsy were screened for CDKL5 mutation using multiplex ligation-dependent probe amplification and DNA sequencing. The clinical sensitivity was reviewed by combining the results of studies using similar inclusion screening criteria. RESULTS: Thirty children (19 girls and 11 boys) with a median seizure onset of 7 months were screened. Almost a half had infantile spasms and one fifth had stereotypic hand movements. A novel c.2854C>T (p.R952X) was identified in an ambulatory girl who had severe mental retardation, multiple types of seizures without Rett-like features. Her mother had a mild intellectual disability, yet her grandmother and half sister were normal despite having the same genetic alteration (random X-inactivation patterns). The pathogenicity of p.R952X identified here was uncertain since healthy relatives and 6 female controls also harbor this alteration. The clinical sensitivity of CDKL5 mutation screening among females with Rett-like features and negative MECP2 screening was 7.8% while the clinical sensitivity among females having cryptogenic intractable seizures with an onset before the ages of 12, 6 and 3 months were 4.7, 11.6 and 14.3%, respectively.