Objective: Single gene mutations leading to severe obesity have so far been identified in 3-5% cases in European populations. However, prevalence of these pathogenic mutations has not systematically been examined in specific consanguineous populations. Here we describe the incidence of obesity-associated mutations through a step-wise sequence analysis, in a cohort of 73 Pakistani children with severe obesity from consanguineous families. Methods: Initially, all subjects were screened for mutations in coding regions of leptin (LEP) and melanocortin 4 receptor (MC4R) genes by direct sequencing. Subjects negative for mutation in these genes were screened using microdroplet PCR enrichment and NGS. Genomic structural variation was assessed by genotyping. Serum leptin, insulin, and cortisol were determined by ELISA. Results: Among 73 children with severe obesity (BMI SDS > 3.0), we identified 22 probands and 5 relatives, carrying 10 different loss-of-function homozygous mutations in LEP, leptin receptor (LEPR), and MC4R genes, including 4 novel variants. Hypercortisolemia was significantly emphasized in LEP mutation carriers. Conclusions: The prevalence of pathogenic mutations in genes known to directly influence leptinmelanocortin signaling is 30% in our cohort. The results of this study emphasize the desirability of undertaking systematic and in-depth genetic analysis of cases with severe obesity in specific consanguineous populations.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

The triploid genome of plantain (Musa spp., AA group) has been considered intractable to genetic improvement because of the production of putatively homogeneous 2n (= 3x) gametes. Plantain breeding schemes have been based on the hybridization of 2n megaspores from the triploid plantain and haploid microspores from a wild diploid banana. The resultant fuli-sib populations of tetrapioid hybrids exhibited extreme variation in phenotypic characteristics. This study was conducted to analyze the genetic constitution of tetraploid hybrids in order to characterize the nature of the gametes from which they were derived. Parental genotypes were screened with 31 primer pairs specific to different Musa microsatcllite loci. Primers which detected polymorphisms between the parental genotypes were used to screen a population of 14 full-sib tetraploid hybrids. A subset of primers was also used to screen a population generated by self-pollination of the parental diploid banana. During analysis of the tetraploid hybrid population, 75% of the alleles donated by the maternal genotype (Obino I'Ewai) were observed to segregate. These data demonstrate the occurrence of recombination during the formation of 2n megaspores in triploid plantain. Analysis of the paternal diploid banana accession (Calcutta 4) and its selfed progeny suggests that this accession is highly heterozygous. These data demonstrate the importance of genetic characterization to Musa breeding, and also indicate that microsatellite markers are well suited for marker-assisted selection systems in Musa. p ILar~TA~N, a cultivated triploid (2n=3x=33) giant perennial herb, is a natural interspecific hybrid between the two wild species, M. acuminata Colla. and M. balbisiana Colla., which provided the A and B genomes, respectively (Simmonds and Shepherd, 1955). The crop is now among the most important staple food crops in the tropical humid forest regions of Africa and South America (FAO, 1994). Plantain has been considered intractable to genetic improvement (DeLanghe, 1976; Rowe, 1984). However, this hypothesis has recently been challenged by the identification of several hundred hybrids from crosses between fertile triploid plantain (AAB) landraces and wild diploid accessions of M. acuminata (AA). The generation of tetraploid Musa hybrids at IITA followed a breeding scheme based on ploidy manipulation. Haploid gametes were donated by the wild banana M. acuminata ssp. burmannica clone Calcutta 4, and 2n (=3x) gametes were donated by a triploid plantain landrace. Crosses between maternal triploid and paternal diploid

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