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Supplementary Table S2. Father's folate pathway genotypes and risk of ALL by immunophenotype and cytogenetic subtype.

Background: Several studies suggest that maternal folic acid supplementation before or during pregnancy protects against childhood acute lymphoblastic leukemia (ALL). We investigated associations between ALL risk and folate pathway gene polymorphisms, and their modification by maternal folic acid supplements, in a population-based case–control study (2003–2007).Methods: All Australian pediatric oncology centers provided cases; controls were recruited by national random digit dialing. Data from 392 cases and 535 controls were included. Seven folate pathway gene polymorphisms (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756 A>G, MTR 5049 C>A, CBS 844 Ins68, and CBS 2199 T>C) were genotyped in children and their parents. Information on prepregnancy maternal folic acid supplement use was collected. ORs were estimated with unconditional logistic regression adjusted for frequency-matched variables and potential confounders. Case–parent trios were also analyzed.Results: There was some evidence of a reduced risk of ALL among children who had, or whose father had, the MTRR 66GG genotype: ORs 0.60 [95% confidence interval (CI) 0.39–0.91] and 0.64 (95% CI, 0.40–1.03), respectively. The ORs for paternal MTHFR 677CT and TT genotypes were 1.41 (95% CI, 1.02–1.93) and 1.81 (95% CI, 1.06–3.07). ORs varied little by maternal folic acid supplementation.Conclusions: Some folate pathway gene polymorphisms in the child or a parent may influence ALL risk. While biologically plausible, underlying mechanisms for these associations need further elucidation.Impact: Folate pathway polymorphisms may be related to risk of childhood ALL, but larger studies are needed for conclusive results. Cancer Epidemiol Biomarkers Prev; 24(1); 48–56. ©2014 AACR.

Supplementary Figure S1. Flow chart of data collection for the folate pathway genotype analysis in the Australian Study of the causes of Acute Lymphoblastic Leukemia in children (Aus-ALL)

Supplemental Tables 1-9. Supplemental Table S1: Tests for deviation from Hardy-Weinberg Equilibrium using parental genotypes. Supplemental Table S2: Folate pathway genotype and the risk of CBT with only cases also available for case-parent trio analysis (N=246). Supplemental Table S3: Folate pathway genotype and the risk of CBT with adjustment only for matching variables. Supplemental Table S4: Folate pathway genotype and the risk of CBT where child has 3 or more European grandparents. Supplemental Table S5: Folate-pathway genotype and the risk of CBT (excluding FTA samples). Supplemental Table S6: Child's folate pathway genotype and risk of CBT by tumor subtype. Supplemental Table S7: Maternal folate pathway genotype and risk of CBT by tumor subtype. Supplemental Table S8: Paternal folate pathway genotype and risk of CBT by tumor subtype. Supplemental Table S9: Child's folate pathway genotype and risk of CBT by sex of the child.

Background: Recent research suggests that maternal folic acid supplementation is associated with a reduced risk of childhood brain tumors (CBT); polymorphisms in folate pathway genes could modify this association or directly influence CBT risk.Methods: Associations between risk of CBT and folate pathway polymorphisms were investigated in a population-based case–control study in Australia (2005–2010). Cases were recruited through all Australian pediatric oncology centers and controls by national random digit dialing. Data were available from 321 cases and 552 controls. Six polymorphisms were genotyped in children and parents (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, MTR 5049C>A, and CBS 2199 T>C). Maternal folic acid use was ascertained via questionnaire. ORs were estimated using unconditional logistic regression. Case–parent trio analyses were also undertaken.Results: There was weak evidence of a reduced risk of CBT for the MTRR 66GG genotype in the child or father: ORs 0.71 [95% confidence interval (CI), 0.48–1.07]; 0.54 (95% CI, 0.34–0.87), respectively. Maternal prepregnancy folic acid supplementation showed a stronger negative association with CBT risk where the child, mother, or father had the MTRR 66GG genotype (Pinteraction = 0.07, 0.10, and 0.18, respectively).Conclusions: Evidence for an association between folate pathway genotypes and CBT is limited in this study. There was possible protection by the MTRR 66GG genotype, particularly when combined with maternal prepregnancy folic acid supplementation; these results are novel and require replication.Impact: The possible interaction between folic acid supplementation and MTRR 66A>G, if confirmed, would strengthen evidence for prepregnancy folate protection against CBT. Cancer Epidemiol Biomarkers Prev; 24(6); 931–7. ©2015 AACR.

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., Genome-wide analysis identifies common noncoding putative regulatory, as well as common and rare potentially deleterious variants directly influencing protein coding, in genes and pathways related to cilium structure and function that may contribute to severe otitis media in Aboriginal Australians.

PurposeThe aim of the study was to examine whether otitis media (OM) in early childhood has an impact on language development in later childhood.MethodsWe 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.ResultsThere was no significant relationship between bilateral OM and language ability at 6 years of age (β = −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 (β = −3.17 [−6.04, −0.31],p =.030), but not for the combined unilateral or bilateral OM group (β = −1.83 [−4.04, 0.39],p =.106).ConclusionsChildren 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.

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.

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.

Summary 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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12866-018-1154-3) contains supplementary material, which is available to authorized users.

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 (

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.

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.

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.

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.

To examine the relationship between early life episodes of otitis media and later behavioural development with adjustment for confounders.Longitudinal cohort study.The Western Australian Pregnancy Cohort (Raine) Study recruited 2900 pregnant women from King Edward Memorial Hospital (KEMH) in Perth, Western Australia, between 1989 and 1991.Data from the children born were collected at both the Year 3 and Year 5 follow-up. At Year 3, n = 611 were diagnosed with recurrent otitis media through parent-report and clinical examination. At Year 5, n = 299 were considered exposed to otitis media based upon tympanometry results.Performance in the Child Behaviour Checklist (CBCL), a questionnaire completed by the primary caregiver at Year 10.Significant associations were found between recurrent otitis media at Year 3 and internalising behaviours (P = .011), and the somatic (P = .011), withdrawn (P = .014), attention (P = .003) and thought problems domains (P = .021), and the total CBCL score (P = .010). A significant association was also found between exposure to otitis media at Year 5 and externalising behaviours (P = .026).A modest association was seen between recurrent otitis media at Year 3 and exposure to otitis media at Year 5 and a number of behaviour domains at Year 10.

Figure S3. Beta diversity PCoA in the nasopharynx of cases and controls, sorted by other covariates. Case and control nasopharyngeal samples shown in Fig. 3 are coloured by other covariates. NA refers to samples where the covariate was not applicable or was missing (not given or recorded â unknownâ ) and the number represents individual samples. (PDF 564 kb)

Figure S1. Diagrammatic overview of the 16S rRNA gene data analysis pipeline. Names of the software or tools used are in red. The SILVA database replaced the default taxonomy database in QIIME (GreenGenes) as GreenGenes 13_8 version does not discriminate between Alloiococcus and Dolosigranulum. (PDF 366 kb)

Background: Male genital anomalies, hypospadias and undescended testis (UDT) have been linked to adult male reproductive disorders, testicular cancer (TC) and decreased fertility. Few population-based studies have evaluated effects on adult fertility outcomes and, in the case of UDT, the importance of early corrective surgery (orchidopexy). Methods: We conducted a population-based cohort study of all liveborn males in Western Australia, 1970-1999 and followed them up until 2016 via data linkage to hospital admission, congenital anomaly, cancer and assisted reproductive technology (ART) registries. Study factors were hypospadias or UDT and study outcomes as TC, paternity and use of ART for male infertility. Cox regression was used to estimate the association (Hazards Ratio, HR) between genital anomalies and paternity; and Log-Binomial regression (Relative Risk, RR) for ART. Findings: The cohort comprised 350,835 males, 2,484 (0*7%) had hypospadias and 7,499 (2*1%) UDT. There were 530 (0*1%) TC cases, 109,544 (31%) men fathered children (paternity) and 2,680 (0*8%) men had ART. UDT was associated with a 2*4-fold increase in TC (HR 2*43; 95% CI 1*65-3*58) and hypospadias with a small but imprecise increase in TC (HR 1*37; 95%CI 0*51-3*67). Both hypospadias and UDT were associated with a 21% reduction in paternity (adjusted HR (aHR): 0*79; 95%CI 0*71-0*89 and aHR 0*79; 95%CI 0*74-0*85, respectively). UDT was associated with a 2-fold increased use of ART (aRR 2*26; 95% CI 1*58-3*25). For every six-months of delayed orchidopexy, there was a 6% increase in risk of TC (HR 1*06; 95%CI: 1*03-1*08), 5% increase in future ART use (HR 1*05; 95%CI 1*03-1*08); and 1% reduction in paternity (RR 0*99; 95%CI: 0*98-0*99). Interpretation: UDT is associated with an increased risk of TC, male infertility and decreased paternity. We provide new evidence to support current guidelines for orchidopexy before 18 months, to decrease the risk of future TC and infertility. Funding Information: National Health and Medical Research Council Competing Interest Declaration: The authors have no conflicts of interest relevant to this article to disclose. Ethical Approval Statement: Ethics approval was obtained from the Human Research Ethics Committee of the Department of Health WA (#2015/36) and the WA Reproductive Technology Council.

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.

Background: Recent research suggests that maternal folic acid supplementation is associated with a reduced risk of childhood brain tumors (CBT); polymorphisms in folate pathway genes could modify this association or directly influence CBT risk. Methods: Associations between risk of CBT and folate pathway polymorphisms were investigated in a population-based case–control study in Australia (2005–2010). Cases were recruited through all Australian pediatric oncology centers and controls by national random digit dialing. Data were available from 321 cases and 552 controls. Six polymorphisms were genotyped in children and parents (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, MTR 5049C>A, and CBS 2199 T>C). Maternal folic acid use was ascertained via questionnaire. ORs were estimated using unconditional logistic regression. Case–parent trio analyses were also undertaken. Results: There was weak evidence of a reduced risk of CBT for the MTRR 66GG genotype in the child or father: ORs 0.71 [95% confidence interval (CI), 0.48–1.07]; 0.54 (95% CI, 0.34–0.87), respectively. Maternal prepregnancy folic acid supplementation showed a stronger negative association with CBT risk where the child, mother, or father had the MTRR 66GG genotype (Pinteraction = 0.07, 0.10, and 0.18, respectively). Conclusions: Evidence for an association between folate pathway genotypes and CBT is limited in this study. There was possible protection by the MTRR 66GG genotype, particularly when combined with maternal prepregnancy folic acid supplementation; these results are novel and require replication. Impact: The possible interaction between folic acid supplementation and MTRR 66A>G, if confirmed, would strengthen evidence for prepregnancy folate protection against CBT. Cancer Epidemiol Biomarkers Prev; 24(6); 931–7. ©2015 AACR.

Background: Several studies suggest that maternal folic acid supplementation before or during pregnancy protects against childhood acute lymphoblastic leukemia (ALL). We investigated associations between ALL risk and folate pathway gene polymorphisms, and their modification by maternal folic acid supplements, in a population-based case–control study (2003–2007). Methods: All Australian pediatric oncology centers provided cases; controls were recruited by national random digit dialing. Data from 392 cases and 535 controls were included. Seven folate pathway gene polymorphisms (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756 A>G, MTR 5049 C>A, CBS 844 Ins68, and CBS 2199 T>C) were genotyped in children and their parents. Information on prepregnancy maternal folic acid supplement use was collected. ORs were estimated with unconditional logistic regression adjusted for frequency-matched variables and potential confounders. Case–parent trios were also analyzed. Results: There was some evidence of a reduced risk of ALL among children who had, or whose father had, the MTRR 66GG genotype: ORs 0.60 [95% confidence interval (CI) 0.39–0.91] and 0.64 (95% CI, 0.40–1.03), respectively. The ORs for paternal MTHFR 677CT and TT genotypes were 1.41 (95% CI, 1.02–1.93) and 1.81 (95% CI, 1.06–3.07). ORs varied little by maternal folic acid supplementation. Conclusions: Some folate pathway gene polymorphisms in the child or a parent may influence ALL risk. While biologically plausible, underlying mechanisms for these associations need further elucidation. Impact: Folate pathway polymorphisms may be related to risk of childhood ALL, but larger studies are needed for conclusive results. Cancer Epidemiol Biomarkers Prev; 24(1); 48–56. ©2014 AACR.

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.

Aim: To describe the prevalence and risk factors of recurrent otitis media (rOM) in an urban Australian population at 3 years of age. Methods: Cross-sectional examination of prevalence and risk factors of rOM in 2280 participants from the Raine Study enrolled from public and private hospitals in Perth, Western Australia, between 1989 and 1991. Parental report questionnaires at 3 years of age were used for rOM identification, with secondary confirmation by otoscopic examination at 1, 2 or 3 years of age. Results: The prevalence of parent-reported rOM was 26.8% (611/2280) and 5.5% (125/2280) for severe rOM in the Study. Independent associations were found between rOM and the presence of older siblings, attendance at day care and the introduction of other milk products at ≤4 months of age. Independent associations for severe rOM were the presence of allergies and attendance at day care. Conclusions: Prevalence rates of rOM within the Raine Study children are similar to a number of other known cohorts. Parity, presence of allergies, attendance at day care and introduction of other milk products at ≤4 months are highlighted as specific risk factors for rOM in this population and presence of allergies and attendance at day care being risk factors for severe rOM. Diagnosis of rOM by parent report and the delay between data collection and reporting are limitations of this study. However, as there is very limited data on OM in urban, non-Indigenous Australian children, this study improves our understanding of OM for this group.

American cutaneous leishmaniasis (ACL) is a vector-transmitted infectious disease with an estimated 1.5 million new cases per year. In Brazil, ACL represents a significant public health problem, with approximately 30,000 new reported cases annually, representing an incidence of 18.5 cases per 100,000 inhabitants. Corte de Pedra is in a region endemic for ACL in the state of Bahia (BA), northeastern Brazil, with 500-1,300 patients treated annually. Over the last decade, population and family-based candidate gene studies were conducted in Corte de Pedra, founded on previous knowledge from studies on mice and humans. Notwithstanding limitations related to sample size and power, these studies contribute important genetic biomarkers that identify novel pathways of disease pathogenesis and possible new therapeutic targets. The present paper is a narrative review about ACL immunogenetics in BA, highlighting in particular the interacting roles of the wound healing gene FLI1 with interleukin-6 and genes SMAD2 and SMAD3 of the transforming growth factor beta signalling pathway. This research highlights the need for well-powered genetic and functional studies on Leishmania braziliensis infection as essential to define and validate the role of host genes in determining resistance/susceptibility regarding this disease.

Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs) (rs2517956, rs2952156, rs1058808) were genotyped in 72 families (208 cases; 372 individuals) from the state of Para (PA). All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR) = 2.22; 95% confidence interval (CI) 1.37-3.59; p = 0.001]. Lepromatous (LL) (OR = 3.25; 95% CI 1.37-7.70; p = 0.007) and tuberculoid (TT) (OR = 1.79; 95% CI 1.04-3.05; p = 0.034) leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808) were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN) and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

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.

Summary Twenty percent of people aged 20 to 79 have type 2 diabetes (T2D) in the United Arab Emirates (UAE). Genome-wide association studies (GWAS) to identify genes for T2D have not been reported for Arab countries. We performed a discovery GWAS in an extended UAE family (N = 178; 66 diabetic; 112 healthy) genotyped on the Illumina Human 660 Quad Beadchip, with independent replication of top hits in 116 cases and 199 controls. Power to achieve genome-wide significance (commonly P = 5 × 10−8) was therefore limited. Nevertheless, transmission disequilibrium testing in FBAT identified top hits at Chromosome 4p12-p13 (KCTD8: rs4407541, P = 9.70 × 10−6; GABRB1: rs10517178/rs1372491, P = 4.19 × 10−6) and 14q13 (PRKD1: rs10144903, 3.92 × 10−6), supported by analysis using a linear mixed model approximation in GenABEL (4p12-p13 GABRG1/GABRA2: rs7662743, Padj-agesex = 2.06 × 10−5; KCTD8: rs4407541, Padj-agesex = 1.42 × 10−4; GABRB1: rs10517178/rs1372491, Padj-agesex = 0.027; 14q13 PRKD1: rs10144903, Padj-agesex = 6.95 × 10−5). SNPs across GABRG1/GABRA2 did not replicate, whereas more proximal SNPs rs7679715 (Padj-agesex = 0.030) and rs2055942 (Padj-agesex = 0.022) at COX7B2/GABRA4 did, in addition to a trend distally at KCTD8 (rs4695718: Padj-agesex = 0.096). Modelling of discovery and replication data support independent signals at GABRA4 (rs2055942: Padj-agesex-combined = 3 × 10−4) and at KCTD8 (rs4695718: Padj-agesex-combined = 2 × 10−4). Replication was observed for PRKD1 rs1953722 (proxy for rs10144903; Padj-agesex = 0.031; Padj-agesex-combined = 2 × 10−4). These genes may provide important functional leads in understanding disease pathogenesis in this population.

Otitis media (OM) is a common disease in early childhood characterised by inflammation of the middle ear. Susceptibility to recurrent acute OM (rAOM; ⩾3 episodes AOM in 6 months) and chronic OM with effusion (COME; middle ear effusion ⩾3 months) is 40–70% heritable. Three bacterial pathogens commonly associated with OM, Streptococcus pneumoniae (Sp), non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mc), have been observed within adenoids and as facultative intracellular pathogens that invade and survive in mononuclear cells. Case/pseudo-control conditional logistic regression analysis of variants in the SLC11A1 gene, initially identified for its role in resistance to intra-macrophage pathogens in mice, revealed association with OM at four polymorphisms ( P best = 0.025) in 531 families (660 affected children) from the Western Australian Family Study of Otitis Media. This included association at the functional promoter GTn polymorphism (rs34448891) with alleles that regulate high (allele 3; odds ratio = 1.2, 95% CI 1.00–1.44, P = 0.04) versus low (allele 2; odds ratio = 0.83, 95% CI 0.69–0.99, P = 0.04) SLC11A1 expression. Haplotype and stepwise conditional logistic regression analyses support a single genetic effect in the proximal region of SLC11A1 , with the haplotype 3_C_C_G across rs34448891_rs2276631_rs3731865_rs2695343 significantly ( P = 0.008) over-transmitted to affected offspring. Stratified analysis showed no association with OM in children who had undergone adenoidectomy (296 children), whereas children with adenoids intact (364 children) showed improved significance at the GTn polymorphism (allele 3: odds ratio = 1.38, 95% CI = 1.10–1.75, P = 0.006). Quantitative RT/PCR demonstrated high expression of SLC11A1 in mononuclear cells isolated from adenoid tissue, with a trend for decreased expression with increasing copies of GTn allele 2. Expression of SLC11A1 was enhanced at 12 ( P = 1.2 × 10 −3 ) and 24 h (P −4 ) after infection of Mono-Mac-6 cells with NTHi. This study identifies SLC11A1 as a novel candidate for OM susceptibility, particularly in children with adenoids intact. Further analysis in other cohorts is required to validate these observations.

Summary Collection of saliva for DNA extraction has created new opportunities to recruit participants from the community for genetic association studies. However, sample return rates are variable. No prior study has specifically addressed how study design impacts sample return. Using data from three large-scale genetic association studies we compared recruitment strategy and sample return rates. We found highly significant differences in sample return rates between the studies. In studies that recruited retrospectively, overall returns were much lower from families with a self-limiting condition who provided samples at a research centre or home visit, than adult elderly individuals with a chronic disease who provided samples by post (59% vs. 84%). Prospective recruitment was associated with high agreement to participate (72%), but subsequent low return of actual saliva samples (42%). A telephone call had marginal effect on recruitment in a retrospective family study, but significantly improved returns in a prospective family study. We found no effect upon DNA yield comparing observed versus unobserved sample collection, or between male and female adult participants. Overall, study design significantly impacts upon response rates for genetic association studies recruiting from the community. Our findings will help researchers in constructing and costing a recruitment protocol.

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.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 (10We 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%; p0.001) or UDT (n = 105; 12.0%; p0.001) 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).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.

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 (

The STAT inhibitor, SOCS1 is reduced in IPF lung fibroblasts, and has been linked to higher levels of type I collagen. Using IPF (n=6) and control (n=9) lung fibroblast cultures and control (n=4) and IPF (n=8) lung tissue, we investigated the mechanisms underlying SOCS1 down-regulation in IPF. We have demonstrated a significant reduction in basal SOCS1 mRNA in IPF fibroblasts and have observed a trend towards reduced SOCS3. Immunohistochemical analysis of lung tissue revealed an abundance of activated-STAT1 and -STAT3, target molecules of SOCS1 and SOCS3. There was no difference in collagen mRNA between IPF and control fibroblasts but high basal levels of collagen protein were detected in IPF cells. There was no difference in the ability of IPF fibroblasts to express pSTAT1, pSTAT3 or SOCS3 following IL-6 stimulation, although the magnitude of the response varied. Similarly, the kinetics of IFNγ-induced SOCS1 mRNA and pSTAT1 levels were not altered in IPF fibroblasts. SOCS1 has been identified as a target of miR155, so we investigated the capacity of miR155 to regulate SOCS1 in IPF. miR155 levels were significantly increased in IPF lung tissue, providing a possible explanation for reduced SOCS1. However, miR155 was reduced in IPF fibroblasts. Analysis of the methylation pattern at putative STAT1/3 binding sites within the SOCS1 promoter has revealed that these sites are unmethylated. In conclusion SOCS1 expression in human lung fibroblasts is not regulated by methylation at STAT1/3 transcription factor binding sites in the SOCS1 promoter or by miR155. Funding: NHMRC Project Grant# 458703 and Raine Medical Research Foundation Grant-in-aid

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.

Study question Are maternal first trimester levels of serum free-beta hCG associated with the development of hypospadias or undescended testis (UDT) in boys? Summary answer Overall, first trimester maternal levels of serum free-beta hCG are not associated with hypospadias or UDT. However, elevated levels were found in severe phenotypes (proximal hypospadias and bilateral UDT) suggesting an altered pathway of hormonal release in early pregnancy. What is known already Human chorionic gonadotrophin peaks in first trimester of pregnancy stimulating fetal testosterone production, which is key to normal male genital development. Endocrine-disrupting insults early in pregnancy have been associated with increased risk of common genital anomalies in males such as hypospadias and UDT. One plausible etiological pathway is altered release of hCG. Study design, size, duration We conducted a record-linkage study of two separate populations of women attending first trimester aneuploidy screening in two Australian states, New South Wales (NSW) and Western Australia (WA), in 2006-2009 and 2001-2003, respectively. Participants/materials, setting, methods Included were women who gave birth to a singleton live born male infant. There were 12 099 boys from NSW and 10 518 from WA included, of whom 90 and 77 had hypospadias; and 107 and 109 UDT, respectively. Serum levels of free-beta hCG were ascertained from laboratory databases and combined with relevant birth outcomes and congenital anomalies via record linkage of laboratory, birth, congenital anomalies and hospital data. Median and quartile levels of gestational age specific free-beta hCG multiple of the median (MoM) were compared between affected and unaffected boys. Logistic regression was used to evaluate the association between levels of free-beta hCG MoM and hypospadias or UDT, stratified by suspected placental dysfunction and co-existing anomalies. Where relevant, pooled analysis was conducted. Main results and the role of chance There was no difference in median hCG levels amongst women with an infant with hypospadias (NSW = 0.88 MoM, P = 0.83; WA = 0.84 MoM, P = 0.76) or UDT (NSW = 0.89 MoM, P = 0.54; WA = 0.95 MoM, P = 0.95), compared with women with an unaffected boy (NSW = 0.92 MoM; WA = 0.88 MoM). Low ( 75th centile) hCG levels were not associated with hypospadias or UDT, nor when stratifying by suspected placental dysfunction and co-existing anomalies. However, there was a tendency towards high levels for severe types, although confidence intervals were wide. When combining NSW and WA results, high hCG MoM levels (>75th centile) were associated with increased risk of proximal hypospadias (odds ratio (OR) 4.34; 95% CI: 1.08-17.4) and bilateral UDT (OR 2.86; 95% CI: 1.02-8.03). Limitations, reasons for caution There were only small numbers of proximal hypospadias and bilateral UDT in both cohorts and although we conducted pooled analyses, results reported on these should be interpreted with caution. Gestational age by ultrasound may have been inaccurately estimated in small and large for gestational age fetuses affecting hCG MoM calculation in those pregnancies. Despite the reliability of our datasets in identifying adverse pregnancy outcomes, we did not have pathology information to confirm tissue lesions in the placenta and therefore our composite outcome should be considered as a proxy for placental dysfunction. Wider implications of the findings This is one of the largest population-based studies examining the association between maternal first trimester serum levels of free-beta hCG and genital anomalies-hypospadias and UDT; and the first to compare specific phenotypes by severity. Overall, our findings does not support the hypothesis that alteration in maternal hCG levels is associated with the development of male genital anomalies; however, high hCG free-beta levels found in severe types suggest different underlying etiology involving higher production and secretion of hCG. These findings require further exploration and replication. Study funding/competing interests This work was funded by the National Health and Medical Research Council (NHMRC) grant APP1047263. N.N. is supported by a NHMRC Career Development Fellowship APP1067066. C.B. was supported by a NHMRC Principal Research Fellowship #634341. The funding agencies had no role in the design, analysis, interpretation or reporting of the findings. There are no competing interests. Trial registration number Not applicable.

To examine the long-term effects of predominant breastfeeding on incidence of otitis media.Prospective birth cohort study.The West Australian Pregnancy Cohort (Raine) Study recruited 2900 mothers through antenatal clinics at the major tertiary obstetric hospital in Perth, Western Australia, between 1989 and 1992.In total, 2237 children participated in a 6-year cohort follow-up, and a subset of 1344 were given ear and hearing assessments.OM diagnosis at 6 years of age (diagnosed by low-compliance tympanograms, 0-0.1 mmho). This was compared to OM diagnosed at the 3-year cohort follow-up using parent-report measures. Main exposure measures were duration of predominant breastfeeding (defined as the age other milk was introduced) and duration of partial (any) breastfeeding (defined as the age breastfeeding was stopped).There was a significant, independent association between predominant breastfeeding (OR = 1.33 [1.04, 1.69]; P = 0.02) and OM, and breastfeeding duration (OR = 1.35 [1.08, 1.68]; P = 0.01) with OM at 3 years of age. However, at 6 years of age, this relationship was no longer statistically significant (predominant breastfeeding OR = 0.78 [0.48, 1.06]; P = 0.09; duration of breastfeeding, OR = 1.34 [0.81, 2.23]; P = 0.25).Our findings are in line with a number of epidemiological studies which show a positive association between breastfeeding and OM in early childhood. However, the long-term follow-up of these children revealed that by 6 years of age, there was no significant influence of breastfeeding on presence of OM. These results suggest that the protective effect of predominant breastfeeding for at least 6 months does not extend to school-age children, where other social and environmental factors may be stronger predictors of OM.

Viruses, bacteria, and parasites have developed strategies to invade and establish long-term infections in their hosts. They have been shown to manipulate their hosts' processes such as the prevention of apoptosis and suppression of the immune response in order to successfully colonize their hosts. Recently, there is increasing interest in the study of epigenetics during an infection, particularly the changes induced in the hosts' DNA methylome, histone marks, and microRNA profiles. In this chapter, we summarize the literature published about viral-, bacterial-, and parasite-induced changes in the host epigenome. We also discuss some pathogens and the potential of the changes they induce in the host epigenome as epigenetic biomarkers to predict clinical outcomes of infection, such as pathogen-induced cancers, or to differentiate between active or latent infection. This might provide the basis to develop more efficient treatments or complement the diagnostic assays currently used clinically.

Leishmania braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. In the mouse, Fli1 was identified as a gene influencing enhanced wound healing and resistance to CL caused by Leishmania major. Polymorphism at FLI1 is associated with CL caused by L. braziliensis in humans, with an inverse association observed for ML disease. Here we extend the analysis to look at other wound healing genes, including CTGF, TGFB1, TGFBR1/2, SMADS 2/3/4/7 and FLII, all functionally linked along with FLI1 in the TGF beta pathway. Haplotype tagging single nucleotide polymorphisms (tag-SNPs) were genotyped using Taqman technology in 325 nuclear families (652 CL cases; 126 ML cases) from Brazil. Robust case-pseudocontrol (CPC) conditional logistic regression analysis showed associations between CL and SNPs at CTGF (SNP rs6918698; CC genotype; OR 1.67; 95%CI 1.10–2.54; P = 0.016), TGFBR2 (rs1962859; OR 1.50; 95%CI 1.12–1.99; P = 0.005), SMAD2 (rs1792658; OR 1.57; 95%CI 1.04–2.38; P = 0.03), SMAD7 (rs4464148; AA genotype; OR 2.80; 95%CI 1.00–7.87; P = 0.05) and FLII (rs2071242; OR 1.60; 95%CI 1.14–2.24; P = 0.005), and between ML and SNPs at SMAD3 (rs1465841; OR 2.15; 95%CI 1.13–4.07; P = 0.018) and SMAD7 (rs2337107; TT genotype; OR 3.70; 95%CI 1.27–10.7; P = 0.016). Stepwise logistic regression analysis showed that all SNPs associated with CL at FLI1, CTGF, TGFBR2, and FLII showed independent effects from each other, but SNPs at SMAD2 and SMAD7 did not add independent effects to SNPs from other genes. These results suggest that TGFβ signalling via SMAD2 is important in directing events that contribute to CL, whereas signalling via SMAD3 is important in ML. Both are modulated by the inhibitory SMAD7 that acts upstream of SMAD2 and SMAD3 in this signalling pathway. Along with the published FLI1 association, these data further contribute to the hypothesis that wound healing processes are important determinants of pathology associated with cutaneous forms of leishmaniasis.

Otitis media (OM) is a common disease in early childhood characterized by inflammation of the middle ear cavity. Heritability studies suggest that there is a substantial genetic component (40%-70%) to the risk of recurrent acute OM, defined as three or more episodes in 6 months or four or more episodes in a year, or chronic OM with effusion (COME), defined as middle ear fluid for ≥ 3 months. To date, only a handful of the regions/genes underlying this genetic susceptibility have been identified. These include several regions of linkage on chromosome 3p25, 10q22, 10q26, 17q12, and 19q13 identified by two genome-wide linkage scans, which appear to harbor susceptibility loci. Fine mapping of these regions has yet to identify the causative genes. Several candidate genes studies have also been reported, with candidates selected on the basis of a plausible biological role in OM or through OM mouse models. Reviewed in this article, these studies have identified positive association at 21 genes, including FBXO11, TLR4, and TNF, with association at five of these replicated in independent populations. However, these studies have been based on small sample sizes, and it is only recently that well-powered OM cohorts suitable for genome-wide association studies (GWAS) have become available. Results from such GWAS will identify novel genes involved in this complex disease. Identification of the genes that contribute to OM susceptibility in childhood will provide important insights into the biological complexity of this disease that could ultimately contribute to improved preventative and therapeutic strategies to reduce the incidence of this disease.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.