High-Throughput Molecular Analysis of Pseudohypoparathyroidism 1b Patients Reveals Novel Genetic and Epigenetic Defects

Context Patients with pseudohypoparathyroidism type 1b (PHP1b) show disordered imprinting of the maternal GNAS allele or paternal uniparental disomy (UPD). Genetic deletions in STX16 or in upstream exons of GNAS are present in many familial but not sporadic cases. Objective Characterization of epigenetic and genetic defects in patients with PHP1b. Design and Patients DNA from 84 subjects, including 26 subjects with sporadic PHP1b, 27 affected subjects and 17 unaffected and/or obligate gene carriers from 12 PHP1b families, 11 healthy individuals, and 3 subjects with PHP1a was subjected to quantitative pyrosequencing of GNAS differentially methylated regions (DMRs), microarray analysis, and microsatellite haplotype analysis. Setting Academic medical center. Main Outcome Measurements Molecular pathology of PHP1b. Results Healthy subjects, unaffected family members and obligate carriers of paternal PHP1b alleles, and subjects with PHP1a showed normal methylation of all DMRs. All PHP1b subjects showed loss of methylation (LOM) at the exon A/B DMR. Affected members of nine PHP1b kindreds showed LOM only at the exon A/B DMR, which was associated with a 3-kb deletion of STX16 exons 4-6 in seven families and a novel deletion of STX16 and adjacent NEPEPL1 in one family. A novel NESP deletion was found in one of two other families with more extensive methylation defects. One sporadic PHP1b had UPD of 20q, two had 3-kb STX16 deletions, and five had apparent epigenetic mosaicism. Conclusions We found diverse patterns of defective methylation and identified novel or previously known mutations in 9 of 12 PHP1b families.

Mycelial Compatibility Groups and Microsatellite Markers Reveal Genetic Diversity Within and Among Populations of Sunflower Sclerotinia Sclerotiorum in China

The genetic variability and differentiation among 101 sunflower Sclerotinia sclerotiorum isolates collected from four different geographic regions of China were analyzed using mycelial compatibility groupings (MCGs) and microsatellite markers. Twenty three MCGs were identified among all tested isolates. The majority of isolates collected from the same region were grouped in to the same MCGs, indicating less genetic variation of S. sclerotiorum within the same region. But there still have exceptions for some isolates. Also microsatellite marker data revealed that all tested isolates from four geographic populations could be divided into three distinct clusters, isolates from Inner Mongolia and Ninxia regions formed cluster I, isolates from Heilongjiang and Xinjiang formed separate clusters II and III . The percentage of variance within and among different geographic populations was 84.54% and 15.46% respectively and both variances were significantly different from each other (p0.01). Meanwhile, association between the microsatellite haplotype and MCGs was observed but not so significant; majority isolates from the same MCG showed the same haplotype, but certain samples showed different haplotypes, although they belonged to the same MCG. Based on the virulence test results, we also found that MCGs not only represent the genetic variation of tested isolates, but also reflect their pathogenic ability to a certain extent.

Abstract 3454: Fine Mapping Of A Chromosome 16 Locus Associated With Low High-density Lipoprotein Cholesterol Level (HDL-C) In French Canadian Subjects

Low HDL-C is a known independent risk factor for coronary heart disease. Levels of HDL-C are influenced by both genetic and environmental factors. We previously performed a quantitative linkage analysis in two independent studies of the French Canadian founder population: a Quebec-wide study (QUE) consisted of 362 individuals from multigenerational families, and 410 individuals from families of the Saguanay-Lac St-Jean region (SLSJ). The results demonstrated a linkage to the locus 16q23–24 in both studies. This locus has been implicated in previous linkage scans for HDL-C in multiple studies from different populations. All of them resulted in linkage peaks that are less than 12 cM far from our peak, suggesting the existence of one or more genes in this specific locus. We examined the region by SNP fine mapping and used family based association and case-control association analysis. Using families from the QUE study, defining HDL-C <5th percentile (age/gender-matched) as cases, the region was narrowed from 25 cM to 18.1 cM. Affected members from four families share a 2 microsatellite haplotype. SNPs genotyped in this locus allowed us to narrow down the shared haplotype to 4Mb. A case-control association study in the SLSJ study sample (cases <5 percentile HDL-C, controls >40 percentile HDL-C) identified several significant SNPs, one of which was located in the same region as the shared haplotype from the QUE families. Constructing a haplotype of this SNP and a nearby SNP, increased the evidence for association (p =0.016 to 0.0097). The CHST6 gene, located within this region, has been previously identified in macular corneal dystrophy. Because of the presence of occular manifestations with other genes associated with low HDL-C (e.g. LCAT, ApoA1), we sequenced the CHST6 gene and also its homologue CHST5, which is located in the same region. We found three non-synonymous variants in these two genes. One of the variants in the CHST6 gene showed a strong segregation in the four families sharing the microsatellite haplotype. This same variant also demonstrated an odds ratio >2 in the case/control sample, but this was not significant due to the small sample size. Our data present strong evidence for a HDL-C gene on chromosome 16q23–24 that may be related to the CHST6 or CHST5 loci.

Common Origin and Fixation of Plasmodium falciparum dhfr and dhps Mutations Associated with Sulfadoxine-Pyrimethamine Resistance in a Low-Transmission Area in South America

ABSTRACT Recent studies indicated that sensitive parasites could increase in frequency in a population when drugs are removed, suggesting that the life span of affordable antimalarial drugs could be expanded. We studied 97 samples from Bolivar State, Venezuela, an area where sulfadoxine-pyrimethamine (SP) has not been used for 8 years due to its ineffectiveness. We characterized point mutations in two genes that have been implicated in resistance to SP, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps). We also assayed neutral microsatellite markers around the dhfr (chromosome 4) and dhps (chromosome 8) genes and on chromosomes 2 and 3 to track the origin and spread of resistant alleles. We found that drug-resistant SP mutants are fixed in the population. Two genotypes were present in the samples, dhfr(50R/51I/108N) dhps(437G/540E/581G) (90.7%) and dhfr(51I/108N) dhps(437G/581G) (9.3%). We show a single microsatellite haplotype for all of the dhfr and dhps alleles, and the alleles at the microsatellite loci are different from those present in Africa. Thus, in these samples from Venezuela, there is a single origin for both dhfr and dhps SP-resistant alleles, and these alleles originated independently of those characterized from Africa. Furthermore, this is the first report of a “hitchhiking effect” on the genetic variation around dhps due to selection by SP using an extensive set of microsatellite markers. Our results indicate that, in areas where there is limited gene flow, the fixation of drug-resistant parasites in the population is stable, even after drug selection is relaxed.

Independent Evolution of Pyrimethamine Resistance in Plasmodium falciparum Isolates in Melanesia

ABSTRACT Pyrimethamine resistance in Plasmodium falciparum has previously been shown to have emerged once in Southeast Asia, from where it spread to Africa. Pyrimethamine resistance in this parasite is known to be conferred by mutations in the gene encoding dihydrofolate reductase (dhfr). We have analyzed polymorphisms in dhfr as well as microsatellite haplotypes flanking this gene in a total of 285 isolates from different regions of Melanesia (Papua New Guinea, Vanuatu, and the Solomon Islands) and Southeast Asia (Thailand and Cambodia). Nearly all isolates (92%) in Melanesia were shown to carry a dhfr double mutation (CN RN I [underlining indicates the mutation]) at positions 50, 51, 59, 108, and 164, whereas 98% of Southeast Asian isolates were either triple (C IRN I) or quadruple (C IRNL ) mutants. Microsatellite analysis revealed two distinct lineages of dhfr double mutants in Melanesia. One lineage had the same microsatellite haplotype as that previously reported for Southeast Asia and Africa, suggesting the spread of this allele to Melanesia from Southeast Asia. The other lineage had a unique, previously undescribed microsatellite haplotype, indicative of the de novo emergence of pyrimethamine resistance in Melanesia.