Neurofibromatosis type 1 gene mutation analysis using sequence capture and high-throughput sequencing.

Neurofibromatosis type 1 syndrome (NF1) is caused by mutations in the NF1 gene. Availability of new sequencing technology prompted us to search for an alternative method for NF1 mutation analysis. Genomic DNA was isolated from saliva avoiding invasive sampling. The NF1 exons with an additional 50bp of flanking intronic sequences were captured and enriched using the SeqCap EZ Choice Library protocol. The captured DNA was sequenced with the Roche/454 GS Junior system. The mean coverages of the targeted regions were 41x and 74x in 2 separate sets of samples. An NF1 mutation was discovered in 10 out of 16 separate patient samples. Our study provides proof of principle that the sequence capture methodology combined with high-throughput sequencing is applicable to NF1 mutation analysis. Deep intronic mutations may however remain undetectable, and change at the DNA level may not predict the outcome at the mRNA or protein levels.

Large-scale zygosity testing using single nucleotide polymorphisms.

A requirement for performing robust genetic and statistical analyses on twins is correctly assigned zygosities. In order to increase the power to detect small risk factors of disease, zygosity testing should also be amenable for high throughput screening. In this study we validate and implement the use of a panel of 50 single nucleotide polymorphisms (SNPs) for reliable high throughput zygosity testing and compare it to a panel of 16 short tandem repeats (STRs). We genotyped both genomic (gDNA) and whole genome amplified DNA (WGA DNA), ending up with 47 SNP and 11 STR markers fulfilling our quality criteria. Out of 99 studied twin pairs, 2 were assigned a different zygosity using SNP and STR data as compared to self reported zygosity in a questionnaire. We also performed a sensitivity analysis based on simulated data where we evaluated the effects of genotyping error, shifts in allele frequencies and missing data on the qualitative zygosity assignments. The frequency of false positives was less than 0.01 when assuming a 1% genotyping error, a decrease of 10% of the observed minor allele frequency compared to the actual values and up to 10 missing markers. The SNP markers were also successfully genotyped on both gDNA and WGA DNA from whole blood, saliva and filter paper. In conclusion, we validate a robust panel of 47 highly multiplexed SNPs that provide reliable and high quality data on a range of different DNA templates.

Identification of MAMDC1 as a candidate susceptibility gene for systemic lupus erythematosus (SLE).

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiple susceptibility genes. We have previously reported suggestive linkage to the chromosomal region 14q21-q23 in Finnish SLE families. PRINCIPAL FINDINGS: Genetic fine mapping of this region in the same family material, together with a large collection of parent affected trios from UK and two independent case-control cohorts from Finland and Sweden, indicated that a novel uncharacterized gene, MAMDC1 (MAM domain containing glycosylphosphatidylinositol anchor 2, also known as MDGA2, MIM 611128), represents a putative susceptibility gene for SLE. In a combined analysis of the whole dataset, significant evidence of association was detected for the MAMDC1 intronic single nucleotide polymorphisms (SNP) rs961616 (P -value = 0.001, Odds Ratio (OR) = 1.292, 95% CI 1.103-1.513) and rs2297926 (P -value = 0.003, OR = 1.349, 95% CI 1.109-1.640). By Northern blot, real-time PCR (qRT-PCR) and immunohistochemical (IHC) analyses, we show that MAMDC1 is expressed in several tissues and cell types, and that the corresponding mRNA is up-regulated by the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in THP-1 monocytes. Based on its homology to known proteins with similar structure, MAMDC1 appears to be a novel member of the adhesion molecules of the immunoglobulin superfamily (IgCAM), which is involved in cell adhesion, migration, and recruitment to inflammatory sites. Remarkably, some IgCAMs have been shown to interact with ITGAM, the product of another SLE susceptibility gene recently discovered in two independent genome wide association (GWA) scans. SIGNIFICANCE: Further studies focused on MAMDC1 and other molecules involved in these pathways might thus provide new insight into the pathogenesis of SLE.