PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene.

The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.

High prevalence of hearing impairment in HIV-infected Peruvian children.

OBJECTIVES: To measure the prevalence and to identify risk factors of hearing impairment in human immunodeficiency virus-infected children living in Peru. STUDY DESIGN: Cross-sectional observational study. SETTING: Two public hospitals and 1 nonprofit center in Lima, Peru, between August 2009 and April 2010. SUBJECTS: A total of 139 HIV-infected children, ages 4 to 19 years. METHODS: Hearing impairment and otologic health were assessed with pure tone audiometry, tympanometry, and otoscopy. The primary outcome was hearing loss, defined as average threshold >25dB for 0.5, 1, 2, and 4 kHz, in one or both ears. Historical and socioeconomic information was obtained through parental survey and medical chart review. Statistical analysis included univariate analysis and multivariate logistic regression. RESULTS: Fifty-four (38.8%) of 139 children had hearing impairment. On multivariate analysis, risk factors included: tympanic membrane perforation (odds ratio [OR] 7.08; 95% confidence interval [CI], 1.65-30.5; P = .01), abnormal tympanometry (OR 2.71; 95% CI, 1.09-6.75; P = .03), cerebral infection (OR 11.6; 95% CI, 1.06-126; P = .05), seizures (OR 5.20; 95% CI, 1.21-22.4; P = .03), and CD4 cell count <500 cells/mm(3) (OR 3.53; 95% CI, 1.18-10.5; P = .02). CONCLUSIONS: The prevalence of hearing impairment in HIV-infected children in Lima, Peru was 38.8%. Middle ear disease, prior cerebral infection, and low CD4 cell count were significantly associated with hearing impairment. The high prevalence of hearing impairment emphasizes the need for periodic hearing assessment in the routine clinical care of HIV-infected children.