Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

Common and Rare Genetic Variants That Could Contribute to Severe Otitis Media in an Australian Aboriginal Population.

BACKGROUND: Our goal was to identify genetic risk factors for severe otitis media (OM) in Aboriginal Australians. METHODS: Illumina® Omni2.5 BeadChip and imputed data were compared between 21 children with severe OM (multiple episodes chronic suppurative OM and/or perforations or tympanic sclerosis) and 370 individuals without this phenotype, followed by FUnctional Mapping and Annotation (FUMA). Exome data filtered for common (EXaC_all ≥ 0.1) putative deleterious variants influencing protein coding (CADD-scaled scores ≥15] were used to compare 15 severe OM cases with 9 mild cases (single episode of acute OM recorded over ≥3 consecutive years). Rare (ExAC_all ≤ 0.01) such variants were filtered for those present only in severe OM. Enrichr was used to determine enrichment of genes contributing to pathways/processes relevant to OM. RESULTS: FUMA analysis identified 2 plausible genetic risk loci for severe OM: NR3C1 (Pimputed_1000G = 3.62 × 10-6) encoding the glucocorticoid receptor, and NREP (Pimputed_1000G = 3.67 × 10-6) encoding neuronal regeneration-related protein. Exome analysis showed: (i) association of severe OM with variants influencing protein coding (CADD-scaled ≥ 15) in a gene-set (GRXCR1, CDH23, LRP2, FAT4, ARSA, EYA4) enriched for Mammalian Phenotype Level 4 abnormal hair cell stereociliary bundle morphology and related phenotypes; (ii) rare variants influencing protein coding only seen in severe OM provided gene-sets enriched for "abnormal ear" (LMNA, CDH23, LRP2, MYO7A, FGFR1), integrin interactions, transforming growth factor signaling, and cell projection phenotypes including hair cell stereociliary bundles and cilium assembly. CONCLUSIONS: This study highlights interacting genes and pathways related to cilium structure and function that may contribute to extreme susceptibility to OM in Aboriginal Australian children.

A flexible computational pipeline for research analyses of unsolved clinical exome cases.

Exome sequencing has enabled molecular diagnoses for rare disease patients but often with initial diagnostic rates of ~25-30%. Here we develop a robust computational pipeline to rank variants for reassessment of unsolved rare disease patients. A comprehensive web-based patient report is generated in which all deleterious variants can be filtered by gene, variant characteristics, OMIM disease and Phenolyzer scores, and all are annotated with an ACMG classification and links to ClinVar. The pipeline ranked 21/34 previously diagnosed variants as top, with 26 in total ranked ≤7th, 3 ranked ≥13th; 5 failed the pipeline filters. Pathogenic/likely pathogenic variants by ACMG criteria were identified for 22/145 unsolved cases, and a previously undefined candidate disease variant for 27/145. This open access pipeline supports the partnership between clinical and research laboratories to improve the diagnosis of unsolved exomes. It provides a flexible framework for iterative developments to further improve diagnosis.

Does Otitis Media Affect Later Language Ability? A Prospective Birth Cohort Study.

Purpose The aim of the study was to examine whether otitis media (OM) in early childhood has an impact on language development in later childhood. Methods We analyzed data from 1,344 second-generation (Generation 2) participants in the Raine Study, a longitudinal pregnancy cohort established in Perth, Western Australia, between 1989 and 1991. OM was assessed clinically at 6 years of age. Language development was measured using the Peabody Picture Vocabulary Test-Revised (PPVT-R) at 6 and 10 years of age and the Clinical Evaluation of Language Fundamentals-Third Edition at 10 years of age. Logistic regression analysis accounted for a wide range of social and environmental covariates. Results There was no significant relationship between bilateral OM and language ability at 6 years of age (ß = -0.56 [-3.78, 2.66], p = .732). However, while scores were within the normal range for the outcome measures at both time points, there was a significant reduction in the rate of receptive vocabulary growth at 10 years of age (PPVT-R) for children with bilateral OM at 6 years of age (ß = -3.17 [-6.04, -0.31], p = .030), but not for the combined unilateral or bilateral OM group (ß = -1.83 [-4.04, 0.39], p = .106). Conclusions Children with OM detected at 6 years of age in this cohort had average language development scores within the normal range at 6 and 10 years of age. However, there was a small but statistically significant reduction in the rate of receptive vocabulary growth at 10 years of age (on the PPVT-R measure only) in children who had bilateral OM at 6 years of age after adjusting for a range of sociodemographic factors.

Reviewing the Pathogenic Potential of the Otitis-Associated Bacteria Alloiococcus otitidis and Turicella otitidis.

Alloiococcus otitidis and Turicella otitidis are common bacteria of the human ear. They have frequently been isolated from the middle ear of children with otitis media (OM), though their potential role in this disease remains unclear and confounded due to their presence as commensal inhabitants of the external auditory canal. In this review, we summarize the current literature on these organisms with an emphasis on their role in OM. Much of the literature focuses on the presence and abundance of these organisms, and little work has been done to explore their activity in the middle ear. We find there is currently insufficient evidence available to determine whether these organisms are pathogens, commensals or contribute indirectly to the pathogenesis of OM. However, building on the knowledge currently available, we suggest future approaches aimed at providing stronger evidence to determine whether A. otitidis and T. otitidis are involved in the pathogenesis of OM. Such evidence will increase our understanding of the microbial risk factors contributing to OM and may lead to novel treatment approaches for severe and recurrent disease.

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Association between male genital anomalies and adult male reproductive disorders: a population-based data linkage study spanning more than 40 years.

BACKGROUND: The male genital anomalies hypospadias and undescended testes have been linked to adult male reproductive disorders, testicular cancer, and decreased fertility. Few population-based studies have evaluated their effects on adult fertility outcomes and, in the case of undescended testes, the importance of early corrective surgery (orchidopexy). METHODS: We did a population-based cohort study of all liveborn boys in Western Australia in 1970-99, and followed them up until 2016 via data linkage to registries for hospital admissions, congenital anomalies, cancer, and assisted reproductive technologies (ART). Study factors were hypospadias or undescended testes, and study outcomes were testicular cancer, paternity, and use of ART for male infertility. Cox regression was used to calculate hazard ratios (HRs) for the associations between genital anomalies and testicular cancer or paternity, and log-binomial regression was used to calculate relative risks (RRs) for the associations between genital anomalies and use of ART. FINDINGS: The cohort comprised 350 835 boys, of whom 2484 (0·7%) had been diagnosed with hypospadias and 7499 (2·1%) with undescended testes. There were 505 (0·1%) cases of testicular cancer, 109 471 (31·2%) men had fathered children, and 2682 (0·8%) had undergone fertility treatment with ART. Undescended testes was associated with a more than two times increase in risk of testicular cancer (HR 2·43, 95% CI 1·65-3·58) and hypospadias with an almost 40% increase (1·37, 0·51-3·67), although this increase was not significant. Both hypospadias and undescended testes were associated with a 21% reduction in paternity (adjusted HR 0·79 [95% CI 0·71-0·89] for hypospadias and 0·79 [0·74-0·85] for undescended testes). Undescended testes was associated with a two times increase in use of ART (adjusted RR 2·26, 95% CI 1·58-3·25). For every 6 months' delay in orchidopexy, there was a 6% increase in risk of testicular cancer (HR 1·06, 95% CI 1·03-1·08), a 5% increase in risk of future use of ART (1·05, 1·03-1·08), and a 1% reduction in paternity (RR 0·99, 95% CI 0·98-0·99). INTERPRETATION: Undescended testes is associated with an increased risk of testicular cancer and male infertility, and decreased paternity. We provide new evidence to support current guidelines for orchidopexy before age 18 months to decrease the risk of future testicular cancer and infertility. FUNDING: National Health and Medical Research Council and Sydney Medical School Foundation.

Arylsulphatase A Pseudodeficiency (ARSA-PD), hypertension and chronic renal disease in Aboriginal Australians.

Chronic renal disease (CRD) associated with cardiovascular disease (CVD) and/or type 2 diabetes (T2D) is a significant problem in Aboriginal Australians. Whole exome sequencing data (N = 72) showed enrichment for ClinVar pathogenic variants in gene sets/pathways linking lipoprotein, lipid and glucose metabolism. The top Ingenuity Pathway Analysis canonical pathways were Farsenoid X Receptor and Retinoid Receptor (FXR/RXR; (P = 1.86 × 10-7), Liver X Receptor and Retinoid Receptor (LXR/RXR; P = 2.88 × 10-6), and atherosclerosis signalling (P = 3.80 × 10-6). Top pathways/processes identified using Enrichr included: Reactome 2016 chylomicron-mediated lipid transport (P = 3.55 × 10-7); Wiki 2016 statin (P = 8.29 × 10-8); GO Biological Processes 2017 chylomicron remodelling (P = 1.92 × 10-8). ClinVar arylsulfatase A pseudodeficiency (ARSA-PD) pathogenic variants were common, including the missense variant c.511 G > A (p.Asp171Asn; rs74315466; frequency 0.44) only reported in Polynesians. This variant is in cis with known ARSA-PD 3' regulatory c.*96 A > G (rs6151429; frequency 0.47) and missense c.1055 A > G (p.Asn352Ser; rs2071421; frequency 0.47) variants. These latter two variants are associated with T2D (risk haplotype GG; odds ratio 2.67; 95% CI 2.32-3.08; P = 2.43 × 10-4) in genome-wide association data (N = 402), but are more strongly associated with quantitative traits (DBP, SBP, ACR, eGFR) for hypertension and renal function in non-diabetic than diabetic subgroups. Traits associated with CVD, CRD and T2D in Aboriginal Australians provide novel insight into function of ARSA-PD variants.

Epigenetic dysregulation of host gene expression in Toxoplasma infection with specific reference to dopamine and amyloid pathways.

Recent interest has focussed on the influence of infectious disease organisms on the host epigenome. Toxoplasma gondii infection acquired congenitally or in early life is associated with severe ocular and brain developmental anomalies, while persistent asymptomatic infection is a proposed risk factor for neurodegenerative and psychiatric disorders, including Parkinson's and Alzheimer's Diseases, and schizophrenia. Genome-wide analysis of the host methylome and transcriptome following T. gondii infection in a retinal cell line identified genes (132, 186 and 128 genes at 2, 6 and 24 h post-infection) concordant for methylation and expression, i.e. hypermethylated and decreased expression or hypomethylated and increased expression. Pathway analyses showed perturbation of two neurologically-associated pathways: dopamine-DARPP32 feedback in cAMP signalling (p-value = 8.3 × 10-5; adjusted p-value = 0.020); and amyloid processing (p-value = 1.0 × 10-3; adjusted p-value = 0.043). Amyloid Precursor Protein (APP) decreased in level following T. gondii infection. These results are of interest given the expression of APP early in nervous system development affecting neural migration and the role of amyloid processing in Alzheimer's disease, while dopamine has roles in the developing retina as well as in Parkinson's disease and schizophrenia. Our results provide a possible functional link between T. gondii infection and congenital/early life and adult neurological clinical signs.

An in silico pipeline to filter the Toxoplasma gondii proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation.

Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.

A microbiome case-control study of recurrent acute otitis media identified potentially protective bacterial genera.

BACKGROUND: Recurrent acute otitis media (rAOM, recurrent ear infection) is a common childhood disease caused by bacteria termed otopathogens, for which current treatments have limited effectiveness. Generic probiotic therapies have shown promise, but seem to lack specificity. We hypothesised that healthy children with no history of AOM carry protective commensal bacteria that could be translated into a specific probiotic therapy to break the cycle of re-infection. We characterised the nasopharyngeal microbiome of these children (controls) in comparison to children with rAOM (cases) to identify potentially protective bacteria. As some children with rAOM do not appear to carry any of the known otopathogens, we also hypothesised that characterisation of the middle ear microbiome could identify novel otopathogens, which may also guide the development of more effective therapies. RESULTS: Middle ear fluids, middle ear rinses and ear canal swabs from the cases and nasopharyngeal swabs from both groups underwent 16S rRNA gene sequencing. The nasopharyngeal microbiomes of cases and controls were distinct. We observed a significantly higher abundance of Corynebacterium and Dolosigranulum in the nasopharynx of controls. Alloiococcus, Staphylococcus and Turicella were abundant in the middle ear and ear canal of cases, but were uncommon in the nasopharynx of both groups. Gemella and Neisseria were characteristic of the case nasopharynx, but were not prevalent in the middle ear. CONCLUSIONS: Corynebacterium and Dolosigranulum are characteristic of a healthy nasopharyngeal microbiome. Alloiococcus, Staphylococcus and Turicella are possible novel otopathogens, though their rarity in the nasopharynx and prevalence in the ear canal means that their role as normal aural flora cannot be ruled out. Gemella and Neisseria are unlikely to be novel otopathogens as they do not appear to colonise the middle ear in children with rAOM.

An in silico pipeline to filter the Toxoplasma gondii proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation

Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.

Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.

Early Childhood Development of Boys with Genital Anomalies.

BACKGROUND: Male genital anomalies often require surgery in early life to address functional and cosmetic consequences. However, there has been little assessment of developmental outcomes of affected boys. METHODS: We conducted a population-based cohort study of all boys born in New South Wales, Australia, and undergoing school-entry developmental assessment in 2009 or 2012. Health and developmental information was obtained by means of record-linkage of birth, hospital and Australian Early Development Census data. Boys with hypospadias or undescended testis (UDT) were compared with those without. Developmental outcomes were assessed in five domains (physical health, emotional maturity, communication, cognitive skills, and social competence), and boys were categorized as vulnerable (<10th centile of national scores), developmentally high risk (DHR; vulnerable in 2+ domains), and special needs. RESULTS: We included 420 boys with hypospadias, 873 with UDT, and 77,176 unaffected boys. There was no difference in the proportion of boys developmentally vulnerable in any domain or DHR between boys with hypospadias (DHR: n = 49; 13.1%; p = 0.9), UDT (n = 116; 15.2%; p = 0.06), and unaffected boys (n = 9278; 12.9%). Compared with unaffected boys (n = 4826; 6.3%), boys with hypospadias (n = 43; 10.2%; p < 0.001) or UDT (n = 105; 12.0%; p < 0.001) were more likely to have special needs. Stratified analyses revealed that only boys with UDT and coexisting anomalies had increased risk of being DHR (odds ratio: 2.65; 95% confidence interval, 1.61-4.36) or special needs (odds ratio: 2.91; 95% confidence interval, 2.00-4.22). CONCLUSION: We found no increased risk of poor development among boys with hypospadias or UDT. However, boys with UDT and coexisting anomalies were more likely to have poorer development and special needs. Birth Defects Research 109:535-542, 2017. © 2017 Wiley Periodicals, Inc.

Analysis of expression of FLI1 and MMP1 in American cutaneous leishmaniasis caused by Leishmania braziliensis infection.

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1.

Toxoplasma gondii Infection Is Associated with Mitochondrial Dysfunction in-Vitro.

Upon invasion of host cells, the ubiquitous pathogen Toxoplasma gondii manipulates several host processes, including re-organization of host organelles, to create a replicative niche. Host mitochondrial association to T. gondii parasitophorous vacuoles is rapid and has roles in modulating host immune responses. Here gene expression profiling of T. gondii infected cells reveals enrichment of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial dysfunction 6 h post-infection. We identified 11 hub genes (HIF-1α, CASP8, FN1, POU5F1, CD44, ISG15, HNRNPA1, MDM2, RPL35, VHL, and NUPR1) and 10 predicted upstream regulators, including 4 endogenous regulators RICTOR, KDM5A, RB1, and D-glucose. We characterized a number of mitochondrial parameters in T. gondii infected human foreskin fibroblast cells over a 36 h time-course. In addition to the usual rapid recruitment and apparent enlargement of mitochondria around the parasitophorous vacuole we observed fragmented host mitochondria in infected cells, not linked to cellular apoptosis, from 24 h post-infection. An increase in mitochondrial superoxide levels in T. gondii infected cells was observed that required active parasite invasion and peaked at 30 h post-infection. Measurement of OXPHOS proteins showed decreased expression of Complex IV in infected cells at 24 h post-infection, followed by decreased expression of Complexes I and II at 36 h post-infection. No change occurred in Complex V. No difference in host mitochondrial membrane potential between infected and mock-infected cells was observed at any time. Our results show perturbation of host mitochondrial function following T. gondii infection that likely impacts on pathogenesis of disease.

No simple answers for the Finnish and Russian Karelia allergy contrast: Methylation of CD14 gene.

BACKGROUND: Finnish and Russian Karelian children have a highly contrasting occurrence of asthma and allergy. In these two environments, we studied associations between total serum immunoglobulin E (IgE) with methylation levels in cluster of differentiation 14 (CD14). METHODS: Five hundred Finnish and Russian Karelian children were included in four groups: Finnish children with high IgE (n = 126) and low IgE (n = 124) as well as Russian children with high IgE (n = 125) and low IgE (n = 125). DNA was extracted from whole blood cells and pyrosequenced. Three CpG sites were selected in the promoter region of CD14. RESULTS: Methylation levels in two of the three CpG sites were higher in the Finnish compared to Russian Karelian children. In the promoter area of CD14, the Finnish compared to Russian children with low IgE had a significant (p < 0.0001) increase in methylation levels at the Amp5Site 2. Likewise, the Finnish compared to Russian children with high IgE had a significant (p = 0.003) increase in methylation levels at the Amp5Site 3. In Russian children with low vs. high IgE, there were significant differences in methylation levels, but this was not the case on the Finnish side. In the regression analysis, adding the methylation variation of CD14 to the model did not explain the higher asthma and allergy risk in the Finnish children. CONCLUSIONS: The methylation levels in the promoter region of CD14 gene were higher in the Finnish compared to Russian Karelian children. However, the methylation variation of this candidate gene did not explain the asthma and allergy contrast between these two areas.

Reference genotype and exome data from an Australian Aboriginal population for health-based research.

Genetic analyses, including genome-wide association studies and whole exome sequencing (WES), provide powerful tools for the analysis of complex and rare genetic diseases. To date there are no reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 72 Aboriginal individuals to a depth of 20X coverage in ∼80% of the sequenced nucleotides. We determined 320,976 single nucleotide variants (SNVs) and 47,313 insertions/deletions using the Genome Analysis Toolkit. We had previously genotyped a subset of the Aboriginal individuals (70/72) using the Illumina Omni2.5 BeadChip platform and found ~99% concordance at overlapping sites, which suggests high quality genotyping. Finally, we compared our SNVs to six publicly available variant databases, such as dbSNP and the Exome Sequencing Project, and 70,115 of our SNVs did not overlap any of the single nucleotide polymorphic sites in all the databases. Our data set provides a useful reference point for genomic studies on Aboriginal Australians.

The -308 bp TNF gene polymorphism influences tumor necrosis factor expression in leprosy patients in Bahia State, Brazil.

Leprosy or Hansen's disease is a debilitating chronic granulomatous disease caused by Mycobacterium leprae, with high incidence and prevalence in Brazil. The -308 bp G/A single nucleotide polymorphism (SNP rs1800629) in the tumor necrosis factor (TNF) gene promoter is a proposed risk factor for leprosy. In Brazil, Northern India, Egypt and Nepal, the common G allele was associated with leprosy. In Eastern India, Thailand and Malawi the minor A allele was the risk factor. Allele A was previously associated with high TNF. We genotyped rs1800629 in 326 leprosy cases from Bahia State, Brazil, including 72 paucibacillary (PB) and 47 multibacillary (MB) without reactions, and 69 reversal reaction (RR) and 78 erythema nodosum leprosum (ENL) with reactions. Logistic regression was used to compare patient groups with 331 healthy controls. Relative TNF mRNA was determined in peripheral blood leukocytes by QRTPCR, and serum TNF levels measured by ELISA. We found that TNF mRNA expression was higher (P=0.03) in leprosy patients compared to endemic controls, but did not differ significantly between clinical subgroups. Carriage of the minor A allele was associated (P=0.003) with low TNF mRNA across leprosy patients. Nevertheless, we found no evidence for either allele at this SNP as a risk factor for leprosy per se (OR=1.12, 95% CI 0.79-1.60, P=0.52), PB (OR=0.99, 95% CI 0.54-1.81, P=0.97), MB (OR=0.86, 95% CI 0.40-1.83, P=0.70), RR (OR=1.37, 95% CI 0.79-2.38, P=0.27) or ENL (OR=0.76, 95% CI 0.40-1.45, P=0.42) when compared to endemic controls. Further studies are required to determine whether the influence of the minor A allele on TNF mRNA levels determines response to treatment, particularly in the context of ENL reaction treatment with anti-TNF therapies and RR reactions where treatment with prednisolone is known to reduce TNF levels. Our findings contribute to understanding TNF as an important determinant of leprosy immunopathology in Brazil.

Genetic and environmental factors in the aetiology of hypospadias.

This article reviews the current evidence and knowledge of the aetiology of hypospadias. Hypospadias remains a fascinating anomaly of the male phallus. It may be an isolated occurrence or part of a syndrome or field defect. The increasing use of assisted reproductive techniques and hormonal manipulation during pregnancy may have been associated with an apparent rise in the incidence of hypospadias. Genetic studies and gene analysis have suggested some defects that could result in hypospadias. New light has also been thrown on environmental factors that could modulate candidate genes, causing altered development of the male external genitalia.