Identification of Allelic Polymorphism in the Ovine Leptin Gene

Leptin is an adipocyte-derived hormone/cytokine that influences the physiological control of numerous biological functions and links nutritional status with both neuroendocrine and immune functions. In livestock, variation in the leptin (LEP) gene has been characterized in cattle and pig, but it has not been reported in sheep. In this study, variation in the exon 3 coding sequence of the ovine LEP gene was investigated by polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) analysis and DNA sequencing. Five novel SSCP patterns, representing five different sequences, were identified under a combination of two different electrophoresis conditions. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology with the LEP sequences from a variety of species, suggesting that these sequences represent alleles of the ovine LEP gene. Four single nucleotide polymorphisms (SNPs) were detected, and three of these resulted in amino acid changes. Variation detected here might have an impact on leptin activity and function.     

Polymorphism of the IGHA gene in sheep

Genetic variation in immunoglobulin A, the most abundant immunoglobulin in mammalian cells, has not been reported in ruminants. In this study, variation in the immunoglobulin heavy alpha chain constant gene (IGHA) of sheep was investigated by amplification of a fragment that included the hinge coding sequence, followed by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Three novel sequences, each characterized by unique SSCP banding patterns, were identified. One or two sequences were detected in individual sheep and all the sequences identified shared high homology to the published ovine and bovine IGHA sequences, suggesting that these sequences represent allelic variants of the IGHA gene in sheep. Sequence alignment showed that these sequences differed mainly in the 3′ end of exon 1 and in the coding sequence of the hinge region. There was either a deletion or an insertion of two codons in the hinge coding region in these allelic variants. Codon usage in the hinge coding region was quite different from that in the non-hinge coding regions of the gene, suggesting different evolution of the IGHA hinge sequence. Three novel amino acid sequences of ovine IGHA were also predicted, and variation in these sequences might not only affect antigen recognition but also susceptibility to cleavage by bacterial or parasitic proteases. Nucleotide sequence data reported in this paper have been submitted to the NCBI GenBank nucleotide sequence database and have been assigned the accession nos. AY956424–AY956426.     

Allelic variation of ovine MHC class II DQA1 and DQA2 genes

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1 . Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.     

Association analysis of common variants in FOXO3 with type 2 diabetes in a South Indian Dravidian population

Recent studies have identified common variants in forkhead box O3 gene (FOXO3) to be strongly associated with longevity in different populations. But studies have not been carried out to analyse the role of common variants in FOXO3 with type 2 diabetes. Since type 2 diabetes is an age related disorder and FOXO proteins play an important role in the regulation of metabolism, we studied the role of common variants in FOXO3 for association with type 2 diabetes. The study was carried out in 994 type 2 diabetic samples and 984 normoglycemic control samples from a South Indian Dravidian population. In our analysis, we found that there was no association between any of the selected SNPs in FOXO3 with type 2 diabetes. Analysis of these SNPs with diabetes related biochemical and clinical parameters also did not reveal any significant association. Haplotype association of SNPs in FOXO3 with type 2 diabetes was observed, but the frequency of the haplotypes was considerably lower and they do not remain significant after correction for multiple testing. In conclusion, we did not observe any association of SNPs in FOXO3 with type 2 diabetes and related parameters suggesting an entirely different mechanism by which these SNPs influence longevity. However additional studies in other populations are required to completely rule out the association of common variants in FOXO3 with type 2 diabetes.