Mapping of three novel loci for non-syndromic autosomal recessive mental retardation (NS-ARMR) in consanguineous families from Pakistan.

To date, of 13 loci with linkage to non-syndromic autosomal recessive mental retardation (NS-ARMR), only six genes have been established with associated mutations. Here we present our study on NS-ARMR among the Pakistani population, where people are traditionally bound to marry within the family or the wider clan. In an exceptional, far-reaching genetic survey we have collected more than 50 consanguineous families exhibiting clinical symptoms/phenotypes of NS-ARMR. In the first step, nine families (MR2-9 and MR11) with multiple affected individuals were selected for molecular genetic studies. Two families (MR3, MR4) showed linkage to already know NS-ARMR loci. Fifteen affected and 10 unaffected individuals from six (MR2, MR6, MR7, MR8, MR9 and MR11) families were genotyped by using Affymetrix 5.0 or 6.0 single-nucleotide polymorphism (SNP) microarrays. SNP microarray data was visually inspected by dChip and genome-wide homozygosity analysis was performed by HomozygosityMapper. Additional mapping was performed (to exclude false-positive regions of homozygosity called by HomozygosityMapper and dChip) on all available affected and unaffected members in seven NS-ARMR families, using microsatellite markers. In this manner we were able to map three novel loci in seven different families originating from different areas of Pakistan. Two families (MR2, MR5) showed linkage on chromosome 2p25.3-p25.2. Three families (MR7, MR8, and MR9) that have been collected from the same village and belong to the same clan were mapped on chromosome 9q34.3. MR11 maps to a locus on 9p23-p13.3. Analysis of MR6 showed two positive loci, on chromosome 1q23.2-q23.3 and 8q24.21-q24.23. Genotyping in additional family members has so far narrowed, but not excluded the 1q locus. In summary, through this study we have identified three new loci for NS-ARMR, namely MRT14, 15 and 16.

Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome.

The discovery of an abundance of copy number variants (CNVs; gains and losses of DNA sequences >1 kb) and other structural variants in the human genome is influencing the way research and diagnostic analyses are being designed and interpreted. As such, comprehensive databases with the most relevant information will be critical to fully understand the results and have impact in a diverse range of disciplines ranging from molecular biology to clinical genetics. Here, we describe the development of bioinformatics resources to facilitate these studies. The Database of Genomic Variants (http://projects.tcag.ca/variation/) is a comprehensive catalogue of structural variation in the human genome. The database currently contains 1,267 regions reported to contain copy number variation or inversions in apparently healthy human cases. We describe the current contents of the database and how it can serve as a resource for interpretation of array comparative genomic hybridization (array CGH) and other DNA copy imbalance data. We also present the structure of the database, which was built using a new data modeling methodology termed Cross-Referenced Tables (XRT). This is a generic and easy-to-use platform, which is strong in handling textual data and complex relationships. Web-based presentation tools have been built allowing publication of XRT data to the web immediately along with rapid sharing of files with other databases and genome browsers. We also describe a novel tool named eFISH (electronic fluorescence in situ hybridization) (http://projects.tcag.ca/efish/), a BLAST-based program that was developed to facilitate the choice of appropriate clones for FISH and CGH experiments, as well as interpretation of results in which genomic DNA probes are used in hybridization-based experiments.

Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

SNP association studies in Alzheimer's disease highlight problems for complex disease analysis.

Genetic linkage and association analyses are two distinct approaches to understanding the genetic etiology of complex disease. Association analysis has become particularly popular in recent times, but the true utility of the strategy remains uncertain. To try to gain better insight into the relevant issues, we have used genetic association analysis to explore the etiology of Alzheimer's disease. Our empirical findings supplement the theoretical debate, illustrating the general doubtfulness of previous positive findings and the limited ability of typical association studies based on candidate genes to discern true medium-sized signals from false positives. Improvements in genotyping technologies and increasing the number of SNPs tested, without sophisticated allowance for all other issues, could simply lead to an unmanageable overload of false-positive signals, themselves obscuring true disease associations.

Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

apolipoprotein-E dependent role for the FAS receptor in early onset Alzheimer's disease: finding of a positive association for a polymorphism in the TNFRSF6 gene.

The TNFRSF6 gene encodes FAS, a cell-surface receptor involved in apoptosis initiation. Elevated levels of FAS have been reported in the brains of Alzheimer's disease (AD) patients. We have tested a G/A polymorphism at position -670 in the TNFRSF6 gene for association with non-familial, early onset Alzheimer's disease (EOAD) by using dynamic allele-specific hybridization. In an initial set of Scottish EOAD cases (n=78) and controls (n=152), we found that, for individuals carrying one or two APOE4 alleles, the homozygous GG-genotype was enriched in the patients (26.7% versus 10.9% in controls). A second study was conducted on an independent set of Scottish individuals (87 EOAD, 358 controls). In this material, the TNFRSF6 GG-genotype frequency was elevated in patients regardless of APOE4 status (28.7% versus 15.1%) and was even more enriched in APOE4 carriers (35.9% versus 15.3%). A combination of the two sample sets (165 cases, 510 controls) gave a significant disease association for the TNFRSF6 GG-genotype that was irrespective of APOE4 (P=0.0020) and that was almost completely attributable to the enrichment present within the set of APOE4 carriers (P=0.0016). This represents an odds ratio of 8.71 for GG-homozygotes carrying at least one APOE4 allele compared with other TNFRSF6 genotypes in APOE4 non-carriers. The TNFRSF6 variation was further explored in Scottish late-onset Alzheimer's disease (n=159) but no associations were found. These results imply that TNFRSF6, in interaction with APOE4, is a genetic risk factor for sporadic EOAD. Hence, the AD risk contributed by APOE4 could be mechanistically related to a pathway in common with FAS-mediated apoptosis.