Analysis of the ABCA4 gene by next-generation sequencing.

Invest Ophthalmol Vis Sci. 2011 Sep 12;

Authors: Zernant J, Schubert C, Im KM, Burke T, Brown CM, Fishman GA, Tsang SH, Gouras P, Dean M, Allikmets R

Abstract
PurposeTo find all possible disease-associated variants in coding sequences of the ABCA4 gene in a large cohort of patients diagnosed with ABCA4-associated diseases.MethodsOne hundred and sixty eight patients who had been clinically diagnosed with Stargardt disease, cone-rod dystrophy, and other ABCA4-associated phenotypes, were pre-screened for mutations in ABCA4 with the ABCA4 microarray, resulting in finding 1 out of 2 expected mutations in 111 patients and 0/2 in 57 patients. The next-generation sequencing (NGS) strategy was applied to these patients on the Fluidigm and Roche 454 Titanium platforms. Identified new variants were confirmed or rejected by Sanger sequencing and analyzed for possible pathogenicity by in silico programs and, where possible, by segregation analyses.ResultsSequencing was successful in 159/168 patients and identified the second disease-associated allele in 49/103 (∼48%) of cases with one previously identified mutation. In patients with no mutations, both disease-associated alleles were detected in 4/56 cases and 1 mutation in 10/56 patients. We detected a total of 57 previously unknown possibly pathogenic variants; 29 missense, 4 nonsense, 9 small deletions and 15 splice-site-altering variants. Of these, 55 variants were deemed pathogenic by a combination of predictive methods and segregation analyses.ConclusionsMany mutations in the coding sequences of the ABCA4 gene are still unknown and many possibly reside in non-coding regions of the ABCA4 locus. While the ABCA4 array remains a good first-pass screening option, the NGS platform is a time- and cost-efficient tool for screening large cohorts.

PMID: 21911583 [PubMed - as supplied by publisher]

Targeted resequencing of the EGFR and MET genes using the Fluidigm Access Array System and the Roche GS Junior System.

Biotechniques. 2011 Sep;51(3):198-9

Authors: Teiling C, Pieprzyk M

PMID: 21906044 [PubMed - in process]

Lack of association of NAMPT rs9770242 and rs59744560 polymorphisms with disease susceptibility and cardiovascular risk in patients with rheumatoid arthritis.

Clin Exp Rheumatol. 2011 Jul-Aug;29(4):681-8

Authors: García-Bermúdez M, González-Juanatey C, Rodríguez-Rodríguez L, Miranda-Filloy JA, Perez-Esteban S, Vazquez-Rodriguez TR, Castañeda S, Balsa A, Fernández-Gutierrez B, Llorca J, González-Alvaro I, Martín J, González-Gay MA

Abstract
OBJECTIVES: Visfatin is an adipokine encoded by the NAMPT (PBEF1) gene. In this study we assessed the potential association of two NAMPT gene polymorphisms with disease susceptibility and cardiovascular (CV) risk in patients with rheumatoid arthritis (RA).
METHODS: A total of 1,395 patients fulfilling the 1987 ACR classification criteria for RA and 1,230 matched controls, were genotyped for the NAMPT rs9770242 and rs59744560 gene polymorphisms, located within the proximal promoter, using predesigned TaqMan single nucleotide polymorphism genotyping assay. Also, HLA-DRB1 genotyping was performed using molecular based methods. In a second step, 1,196 patients in whom full information was available were assessed to determine the influence of NAMPT rs9770242 and rs59744560 polymorphisms in the development of CV events. Also, the potential influence of these polymorphisms in the development of subclinical atherosclerosis was assessed in a subgroup of patients with no history of CV events by brachial artery reactivity to determine flow-mediated endothelium-dependent and endothelium-independent vasodilatation (n=125) and by B-mode ultrasonography to determine the carotid artery intima-media thickness (n=105).
RESULTS: No statistically significant differences in the allele or genotype frequencies for the NAMPT gene polymorphisms between RA patients and controls were found. A modest non significant lower frequency of the minor allele G of rs9770242 polymorphism was observed among patients with CV disease (20.62%) compared to those without CV disease (22.83%) (p=0.39). Also, a slight nonsignificant lower frequency of the minor allele T of rs59744560 polymorphism in patients with CV events (9.81%) compared with those RA patients who did not experience CV disease (13.07%) (p=0.11) was observed. Likewise, no significant association between the NAMPT polymorphisms with surrogate markers of subclinical atherosclerosis was found in patients with RA.
CONCLUSIONS: NAMPT rs9770242 and rs59744560 polymorphisms are not markers of disease susceptibility and CV disease in RA.

PMID: 21906432 [PubMed - in process]

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

Genomics. 2011 Aug 28;

Authors: Hoffmann TJ, Zhan Y, Kvale MN, Hesselson SE, Gollub J, Iribarren C, Lu Y, Mei G, Purdy MM, Quesenberry C, Rowell S, Shapero MH, Smethurst D, Somkin CP, Van den Eeden SK, Walter L, Webster T, Whitmer RA, Finn A, Schaefer C, Kwok PY, Risch N

Abstract
Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array (712,930 SNPs) was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.

PMID: 21903159 [PubMed - as supplied by publisher]