High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

Investigation of APOE isoforms and the association between APOE E3 and E4 with migraine in the Australian Caucasian population

Migraine is a debilitating neurovascular disease that is associated with pulsating head pain accompanied by nausea, vomiting, photophobia, phonophobia and sometimes visual sensory disturbances. Because of its role in nitric oxide regulation and interleukin release, apolipoprotein E (APOE) has been suggested to play a role in the migraine pathogenesis pathway. This study evaluated the potential role of three APOE variants in an Australian population and the role that they may play in susceptibility to migraine. The study found no significant association between the tested variants and migraine for any of the APOE variants investigated.

Investigation of lymphotoxin α genetic variants in migraine

Migraine is a common neurological disease with a genetic basis affecting approximately 12% of the population. Pain during a migraine attack is associated with activation of the trigeminal nerve system, which carries pain signals from the meninges and the blood vessels infusing the meninges to the trigeminal nucleus in the brain stem. The release of inflammatory mediators following cortical spreading depression (CSD) may further promote and sustain the activation and sensitization of meningeal nociceptors, inducing the persistent throbbing headache characterised in migraine. Lymphotoxin α (LTA) is a cytokine secreted by lymphocytes and is a member of the tumour necrosis factor (TNF) family. Genetic variation with the TNF and LTA genes may contribute to threshold brain excitability, propagation of neuronal hyperexcitability and thus initiation and maintenance of a migraine attack. Three LTA variants rs2009658, rs2844482 and rs2229094 were identified in a recent pGWAS study conducted in the Norfolk Island population as being potentially implicated in migraine with nominally significant p values of p = 0.0093, p = 0.0088 and p = 0.033 respectively. To determine whether these SNPs played a role in migraine in a general outbred population these SNPs were gentoyped in a large case control Australian Caucasian population and tested for association with migraine. All three SNPs showed no association in our cohort (p > 0.05). Validation of GWAS data in independent case-controls cohorts is essential to establish risk validity within specific population groups. The importance of cytokines in modulating neural inflammation and pain threshold in addition to other studies showing associations between TNF-α and SNPs in the LTA gene with migraine, suggests that LTA could be an important factor contributing to migraine. Although the present study did not support a role for the tested LTA variants in migraine, investigation of other variants within the LTA gene is still warranted.

Perianal disease combined with NOD2 genotype predicts need for IBD-related surgery in Crohn’s Disease patients from a population-based cohort

Background: Patients with Crohn’s disease (CD) often require surgery at some stage of disease course. Prediction of CD outcome is influenced by clinical, environmental, serological, and genetic factors (eg, NOD2). Being able to identify CD patients at high risk of surgical intervention should assist clinicians to decide whether or not to prescribe early aggressive treatment with immunomodulators. Methods: We performed a retrospective analysis of selected clinical (age at diagnosis, perianal disease, active smoking) and genetic (NOD2 genotype) data obtained for a population-based CD cohort from the Canterbury Inflammatory Bowel Disease study. Logistic regression was used to identify predictors of complicated outcome in these CD patients (ie, need for inflammatory bowel disease-related surgery). Results: Perianal disease and the NOD2 genotype were the only independent factors associated with the need for surgery in this patient group (odds ratio=2.84 and 1.60, respectively). By combining the associated NOD2 genotype with perianal disease we generated a single “clinicogenetic” variable. This was strongly associated with increased risk of surgery (odds ratio=3.84, P=0.00, confidence interval, 2.28-6.46) and offered moderate predictive accuracy (positive predictive value=0.62). Approximately 1/3 of surgical outcomes in this population are attributable to the NOD2+PA variable (attributable risk=0.32). Conclusions: Knowledge of perianal disease and NOD2 genotype in patients presenting with CD may offer clinicians some decision-making utility for early diagnosis of complicated CD progression and initiating intensive treatment to avoid surgical intervention. Future studies should investigate combination effects of other genetic, clinical, and environmental factors when attempting to identify predictors of complicated CD outcomes.

Unique X-linked familial FSGS with co-segregating heart block disorder is associated with a mutation in the NXF5 gene

Focal segmental glomerulosclerosis (FSGS) is the consequence of a disease process that attacks the kidney's filtering system, causing serious scarring. More than half of FSGS patients develop chronic kidney failure within 10 years, ultimately requiring dialysis or renal transplantation. There are currently several genes known to cause the hereditary forms of FSGS (ACTN4, TRPC6, CD2AP, INF2, MYO1E and NPHS2). This study involves a large, unique, multigenerational Australian pedigree in which FSGS co-segregates with progressive heart block with apparent X-linked recessive inheritance. Through a classical combined approach of linkage and haplotype analysis, we identified a 21.19 cM interval implicated on the X chromosome. We then used a whole exome sequencing approach to identify two mutated genes, NXF5 and ALG13, which are located within this linkage interval. The two mutations NXF5-R113W and ALG13-T141L segregated perfectly with the disease phenotype in the pedigree and were not found in a large healthy control cohort. Analysis using bioinformatics tools predicted the R113W mutation in the NXF5 gene to be deleterious and cellular studies support a role in the stability and localization of the protein suggesting a causative role of this mutation in these co-morbid disorders. Further studies are now required to determine the functional consequence of these novel mutations to development of FSGS and heart block in this pedigree and to determine whether these mutations have implications for more common forms of these diseases in the general population.

A genome-wide analysis of 'Bounty' descendants implicates several novel variants in migraine susceptibility

Migraine is a common neurological disease with a complex genetic aetiology. The disease affects ~12% of the Caucasian population and females are three times more likely than males to be diagnosed. In an effort to identify loci involved in migraine susceptibility, we performed a pedigree-based genome-wide association study of the isolated population of Norfolk Island, which has a high prevalence of migraine. This unique population originates from a small number of British and Polynesian founders who are descendents of the Bounty mutiny and forms a very large multigenerational pedigree (Bellis et al.; Human Genetics, 124(5):543-5542, 2008). These population genetic features may facilitate disease gene mapping strategies (Peltonen et al.; Nat Rev Genet, 1(3):182-90, 2000. In this study, we identified a high heritability of migraine in the Norfolk Island population (h (2) = 0.53, P = 0.016). We performed a pedigree-based GWAS and utilised a statistical and pathological prioritisation approach to implicate a number of variants in migraine. An SNP located in the zinc finger protein 555 (ZNF555) gene (rs4807347) showed evidence of statistical association in our Norfolk Island pedigree (P = 9.6 × 10(-6)) as well as replication in a large independent and unrelated cohort with >500 migraineurs. In addition, we utilised a biological prioritisation to implicate four SNPs, in within the ADARB2 gene, two SNPs within the GRM7 gene and a single SNP in close proximity to a HTR7 gene. Association of SNPs within these neurotransmitter-related genes suggests a disrupted serotoninergic system that is perhaps specific to the Norfolk Island pedigree, but that might provide clues to understanding migraine more generally.

An X chromosome association scan of the Norfolk Island genetic isolate provides evidence for a novel migraine susceptibility locus at Xq12

Migraine is a common and debilitating neurovascular disorder with a complex envirogenomic aetiology. Numerous studies have demonstrated a preponderance of women affected with migraine and previous pedigree linkage studies in our laboratory have identified susceptibility loci on chromosome Xq24-Xq28. In this study we have used the genetic isolate of Norfolk Island to further analyse the X chromosome for migraine susceptibility loci. An association approach was employed to analyse 14,124 SNPs spanning the entire X chromosome. Genotype data from 288 individuals comprising a large core-pedigree, of which 76 were affected with migraine, were analysed. Although no SNP reached chromosome-wide significance (empirical α = 1×10−5) ranking by P-value revealed two primary clusters of SNPs in the top 25. A 10 SNP cluster represents a novel migraine susceptibility locus at Xq12 whilst a 11 SNP cluster represents a previously identified migraine susceptibility locus at Xq27. The strongest association at Xq12 was seen for rs599958 (OR = 1.75, P = 8.92×10−4), whilst at Xq27 the strongest association was for rs6525667 (OR = 1.53, P = 1.65×10−4). Further analysis of SNPs at these loci was performed in 5,122 migraineurs from the Women’s Genome Health Study and provided additional evidence for association at the novel Xq12 locus (P<0.05). Overall, this study provides evidence for a novel migraine susceptibility locus on Xq12. The strongest effect SNP (rs102834, joint P = 1.63×10−5) is located within the 5′UTR of the HEPH gene, which is involved in iron homeostasis in the brain and may represent a novel pathway for involvement in migraine pathogenesis.

Comparison of genomic DNA extraction techniques from whole blood samples : a time, cost and quality evaluation study

Genomic DNA obtained from patient whole blood samples is a key element for genomic research. Advantages and disadvantages, in terms of time-efficiency, cost-effectiveness and laboratory requirements, of procedures available to isolate nucleic acids need to be considered before choosing any particular method. These characteristics have not been fully evaluated for some laboratory techniques, such as the salting out method for DNA extraction, which has been excluded from comparison in different studies published to date. We compared three different protocols (a traditional salting out method, a modified salting out method and a commercially available kit method) to determine the most cost-effective and time-efficient method to extract DNA. We extracted genomic DNA from whole blood samples obtained from breast cancer patient volunteers and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260/280 ratio and polymerase chain reaction product amplification) of the obtained yield. On average, all three methods showed no statistically significant differences between the final result, but when we accounted for time and cost derived for each method, they showed very significant differences. The modified salting out method resulted in a seven- and twofold reduction in cost compared to the commercial kit and traditional salting out method, respectively and reduced time from 3 days to 1 hour compared to the traditional salting out method. This highlights a modified salting out method as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples.

Complete mitochondrial genome sequencing reveals novel haplotypes in a Polynesian population

The high risk of metabolic disease traits in Polynesians may be partly explained by elevated prevalence of genetic variants involved in energy metabolism. The genetics of Polynesian populations has been shaped by island hoping migration events which have possibly favoured thrifty genes. The aim of this study was to sequence the mitochondrial genome in a group of Maoris in an effort to characterise genome variation in this Polynesian population for use in future disease association studies. We sequenced the complete mitochondrial genomes of 20 non-admixed Maori subjects using Affymetrix technology. DNA diversity analyses showed the Maori group exhibited reduced mitochondrial genome diversity compared to other worldwide populations, which is consistent with historical bottleneck and founder effects. Global phylogenetic analysis positioned these Maori subjects specifically within mitochondrial haplogroup - B4a1a1. Interestingly, we identified several novel variants that collectively form new and unique Maori motifs – B4a1a1c, B4a1a1a3 and B4a1a1a5. Compared to ancestral populations we observed an increased frequency of non-synonymous coding variants of several mitochondrial genes in the Maori group, which may be a result of positive selection and/or genetic drift effects. In conclusion, this study reports the first complete mitochondrial genome sequence data for a Maori population. Overall, these new data reveal novel mitochondrial genome signatures in this Polynesian population and enhance the phylogenetic picture of maternal ancestry in Oceania. The increased frequency of several mitochondrial coding variants makes them good candidates for future studies aimed at assessment of metabolic disease risk in Polynesian populations.

Confirmation that Xq27 and Xq28 are susceptibility loci for migraine in independent pedigrees and a case-control cohort

Investigations into migraine genetics have suggested that susceptibility loci exist on the X chromosome. These reports are supported by evidence that demonstrates male probands as having a higher proportion of affected first-degree relatives as well as the female preponderance of 3:1 that the disorder displays. We have previously implicated the Xq24-28 locus in migraine using two independent multigenerational Australian pedigrees that demonstrated excess allele sharing at the Xq24, Xq27 and Xq28 loci. Here, we expand this work to investigate a further six independent migraine pedigrees using 11 microsatellite markers spanning the Xq27–28 region. Furthermore, 11 candidate genes are investigated in an Australian case-control cohort consisting of 500 cases and 500 controls. Microsatellite analysis showed evidence of excess allele sharing to the Xq27 marker DXS8043 (LOD* 1.38 P = 0.005) in MF879 whilst a second independent pedigree showed excess allele sharing to DXS8061 at Xq28 (LOD* 1.5 P = 0.004). Furthermore, analysis of these key markers in a case control cohort showed significant association to migraine in females at the DXS8043 marker (T1 P = 0.009) and association with MO at DXS8061 (T1 P = 0.05). Further analysis of 11 key genes across these regions showed significant association of a three-marker risk haplotype in the NSDHL gene at Xq28 (P = 0.0082). The results of this study add further support to the presence of migraine susceptibility loci on chromosome Xq27 and Xq28 as well as point to potential candidate genes in the regions that warrant further investigation.

Genotypes of the MTHFR C677T and MTRR A66G genes act independently to reduce migraine disability in response to vitamin supplementation

BACKGROUND: Migraine is a chronic disabling neurovascular condition that may in part be caused by endothelial and cerebrovascular disruption induced by hyperhomocysteinaemia. We have previously provided evidence indicating that reduction of homocysteine by vitamin supplementation can reduce the occurrence of migraine in women. The current study examined the genotypic effects of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene variants on the occurrence of migraine in response to vitamin supplementation. METHODS: This was a 6-month randomized, double-blinded placebo-controlled trial of daily vitamin B supplementation (B(6), B(9) and B(12)) on reduction of homocysteine and of the occurrence of migraine in 206 female patients diagnosed with migraine with aura. RESULTS: Vitamin supplementation significantly reduced homocysteine levels (P<0.001), severity of headache in migraine (P=0.017) and high migraine disability (P=0.022) in migraineurs compared with the placebo effect (P>0.1). When the vitamin-treated group was stratified by genotype, the C allele carriers of the MTHFR C677T variant showed a higher reduction in homocysteine levels (P<0.001), severity of pain in migraine (P=0.01) and percentage of high migraine disability (P=0.009) compared with those with the TT genotypes. Similarly, the A allele carriers of the MTRR A66G variants showed a higher level of reduction in homocysteine levels (P<0.001), severity of pain in migraine (P=0.002) and percentage of high migraine disability (P=0.006) compared with those with the GG genotypes. Genotypic analysis for both genes combined indicated that the treatment effect modification of the MTRR variant was independent of the MTHFR variant. CONCLUSION: This provided further evidence that vitamin supplementation is effective in reducing migraine and also that both MTHFR and MTRR gene variants are acting independently to influence treatment response in female migraineurs.

Heritability and genome-wide linkage analysis of migraine in the genetic isolate of Norfolk Island

Migraine is a common neurovascular disorder with a complex envirogenomic aetiology. In an effort to identify migraine susceptibility genes, we conducted a study of the isolated population of Norfolk Island, Australia. A large portion of the permanent inhabitants of Norfolk Island are descended from 18th Century English sailors involved in the infamous mutiny on the Bounty and their Polynesian consorts. In total, 600 subjects were recruited including a large pedigree of 377 individuals with lineage to the founders. All individuals were phenotyped for migraine using International Classification of Headache Disorders-II criterion. All subjects were genotyped for a genome-wide panel of microsatellite markers. Genotype and phenotype data for the pedigree were analysed using heritability and linkage methods implemented in the programme SOLAR. Follow-up association analysis was performed using the CLUMP programme. A total of 154 migraine cases (25%) were identified indicating the Norfolk Island population is high-risk for migraine. Heritability estimation of the 377-member pedigree indicated a significant genetic component for migraine (h2 = 0.53, P = 0.016). Linkage analysis showed peaks on chromosome 13q33.1 (P = 0.003) and chromosome 9q22.32 (P = 0.008). Association analysis of the key microsatellites in the remaining 223 unrelated Norfolk Island individuals showed evidence of association, which strengthen support for the linkage findings (P ≤ 0.05). In conclusion, a genome-wide linkage analysis and follow-up association analysis of migraine in the genetic isolate of Norfolk Island provided evidence for migraine susceptibility loci on chromosomes 9q22.22 and 13q33.1.

Investigation of homocysteine-pathway-related variants in essential hypertension

Hyperhomocysteinemia (hHcy) has been associated with an increased risk of cardiovascular disease and stroke. Essential hypertension (EH), a polygenic condition, has also been associated with increased risk of cardiovascular related disorders. To investigate the role of the homocysteine (Hcy) metabolism pathway in hypertension we conducted a case-control association study of Hcy pathway gene variants in a cohort of Caucasian hypertensives and age- and sex-matched normotensives. We genotyped two polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR C677T and MTHFR A1298C), one polymorphism in the methionine synthase reductase gene (MTRR A66G), and one polymorphism in the methylenetetrahydrofolate dehydrogenase 1 gene (MTHFD1 G1958A) and assessed their association with hypertension using chi-square analysis. We also performed a multifactor dimensionality reduction (MDR) analysis to investigate any potential epistatic interactions among the four polymorphisms and EH. None of the four polymorphisms was significantly associated with EH and although we found a moderate synergistic interaction between MTHFR A1298C and MTRR A66G, the association of the interaction model with EH was not statistically significant (

Randomised, double blind, placebo-controlled trial of Echinacea supplementation in air travellers

Objective. To identify whether a standardised Echinacea formulation is effective in the prevention of respiratory and other symptoms associated with long-haul flights. Methods. 175 adults participated in a randomised, double-blind placebo-controlled trial travelling back from Australia to America, Europe, or Africa for a period of 1–5 weeks on commercial flights via economy class. Participants took Echinacea (root extract, standardised to 4.4 mg alkylamides) or placebo tablets. Participants were surveyed before, immediately after travel, and at 4 weeks after travel regarding upper respiratory symptoms and travel-related quality of life. Results. Respiratory symptoms for both groups increased significantly during travel (

Role of the apolipoprotein E and catechol-O-methyltransferase genes in prospective and retrospective memory traits

Human memory is a complex neurocognitive process. By combining psychological and molecular genetics expertise, we examined the APOE ε4 allele, a known risk factor for Alzheimer's disease, and the COMT Val 158 polymorphism, previously implicated in schizophrenia, for association with lowered memory functioning in healthy adults. To assess memory type we used a range of memory tests of both retrospective and prospective memory. Genotypes were determined using RFLP analysis and compared with mean memory scores using univariate ANOVAs. Despite a modest sample size (n=197), our study found a significant effect of the APOE ε4 polymorphism in prospective memory. Supporting our hypothesis, a significant difference was demonstrated between genotype groups for means of the Comprehensive Assessment of Prospective Memory total score (p=0.036; ε4 alleles=1.99; all other alleles=1.86). In addition, we demonstrate a significant interactive effect between the APOE ε4 and COMT polymorphisms in semantic memory. This is the first study to investigate both APOE and COMT genotypes in relation to memory in non-pathological adults and provides important information regarding the effect of genetic determinants on human memory.