A flow cytometry-based reporter assay identifies macrolide antibiotics as nonsense mutation read-through agents.

UNLABELLED: A large number of human diseases are caused by nonsense mutations. These mutations result in premature protein termination and the expression of truncated, usually nonfunctional products. A promising therapeutic strategy for patients suffering from premature termination codon (PTC)-mediated disorders is to suppress the nonsense mutation and restore the expression of the affected protein. Such a suppression approach using specific antibiotics and other read-through promoting agents has been shown to suppress PTCs and restore the production of several important proteins. Here, we report the establishment of a novel, rapid, and very efficient method for screening stop-codon read-through agents. We also show that, in both mammalian cells and in a transgenic mouse model, distinct members of the macrolide antibiotic family can induce read-through of disease-causing stop codons leading to re-expression of several key proteins and to reduced disease phenotypes. Taken together, our results may help in the identification and characterization of well-needed customized pharmaceutical PTC suppression agents. KEY MESSAGES: Establishment of a flow cytometry-based reporter assay to identify nonsense mutation read-through agents. Macrolide antibiotics can induce read-through of disease-causing stop codons. Macrolide-induced protein restoration can alleviate disease-like phenotypes.

Dissecting the influence of Neolithic demic diffusion on Indian Y-chromosome pool through J2-M172 haplogroup.

The global distribution of J2-M172 sub-haplogroups has been associated with Neolithic demic diffusion. Two branches of J2-M172, J2a-M410 and J2b-M102 make a considerable part of Y chromosome gene pool of the Indian subcontinent. We investigated the Neolithic contribution of demic dispersal from West to Indian paternal lineages, which majorly consists of haplogroups of Late Pleistocene ancestry. To accomplish this, we have analysed 3023 Y-chromosomes from different ethnic populations, of which 355 belonged to J2-M172. Comparison of our data with worldwide data, including Y-STRs of 1157 individuals and haplogroup frequencies of 6966 individuals, suggested a complex scenario that cannot be explained by a single wave of agricultural expansion from Near East to South Asia. Contrary to the widely accepted elite dominance model, we found a substantial presence of J2a-M410 and J2b-M102 haplogroups in both caste and tribal populations of India. Unlike demic spread in Eurasia, our results advocate a unique, complex and ancient arrival of J2a-M410 and J2b-M102 haplogroups into Indian subcontinent.

LRRK2 and RIPK2 variants in the NOD 2-mediated signaling pathway are associated with susceptibility to Mycobacterium leprae in Indian populations.

In recent years, genome wide association studies have discovered a large number of gene loci that play a functional role in innate and adaptive immune pathways associated with leprosy susceptibility. The immunological control of intracellular bacteria M. leprae is modulated by NOD2-mediated signaling of Th1 responses. In this study, we investigated 211 clinically classified leprosy patients and 230 ethnically matched controls in Indian population by genotyping four variants in NOD2 (rs9302752A/G), LRRK2 (rs1873613A/G), RIPK2 (rs40457A/G and rs42490G/A). The LRRK2 locus is associated with leprosy outcome. The LRRK2 rs1873613A minor allele and respective rs1873613AA genotypes were significantly associated with an increased risk whereas the LRRK2 rs1873613G major allele and rs1873613GG genotypes confer protection in paucibacillary and leprosy patients. The reconstructed GA haplotypes from RIPK2 rs40457A/G and rs42490G/A variants was observed to contribute towards increased risk whereas haplotypes AA was observed to confer protective role. Our results indicate that a possible shared mechanisms underlying the development of these two clinical forms of the disease as hypothesized. Our findings confirm and validates the role of gene variants involved in NOD2-mediated signalling pathways that play a role in immunological control of intracellular bacteria M. leprae.

Strong impact of TGF-ß1 gene polymorphisms on breast cancer risk in Indian women: a case-control and population-based study.

INTRODUCTION: TGF-ß1 is a multi-functional cytokine that plays an important role in breast carcinogenesis. Critical role of TGF-ß1 signaling in breast cancer progression is well documented. Some TGF-ß1 polymorphisms influence its expression; however, their impact on breast cancer risk is not clear. METHODS: We analyzed 1222 samples in a candidate gene-based genetic association study on two distantly located and ethnically divergent case-control groups of Indian women, followed by a population-based genetic epidemiology study analyzing these polymorphisms in other Indian populations. The c.29C>T (Pro10Leu, rs1982073 or rs1800470) and c.74G>C (Arg25Pro, rs1800471) polymorphisms in the TGF-ß1 gene were analyzed using direct DNA sequencing, and peripheral level of TGF-ß1 were measured by ELISA. RESULTS: c.29C>T substitution increased breast cancer risk, irrespective of ethnicity and menopausal status. On the other hand, c.74G>C substitution reduced breast cancer risk significantly in the north Indian group (p = 0.0005) and only in the pre-menopausal women. The protective effect of c.74G>C polymorphism may be ethnicity-specific, as no association was seen in south Indian group. The polymorphic status of c.29C>T was comparable among Indo-Europeans, Dravidians, and Tibeto-Burmans. Interestingly, we found that Tibeto-Burmans lack polymorphism at c.74G>C locus as true for the Chinese populations. However, the Brahmins of Nepal (Indo-Europeans) showed polymorphism in 2.08% of alleles. Mean TGF-ß1 was significantly elevated in patients in comparison to controls (p<0.001). CONCLUSION: c.29C>T and c.74G>C polymorphisms in the TGF-ß1 gene significantly affect breast cancer risk, which correlates with elevated TGF-ß1 level in the patients. The c.29C>T locus is polymorphic across ethnically different populations, but c.74G>C locus is monomorphic in Tibeto-Burmans and polymorphic in other Indian populations.