The Australian Reproductive Genetic Carrier Screening Project (Mackenzie's Mission): Design and Implementation.

Reproductive genetic carrier screening (RGCS) provides people with information about their chance of having children with autosomal recessive or X-linked genetic conditions, enabling informed reproductive decision-making. RGCS is recommended to be offered to all couples during preconception or in early pregnancy. However, cost and a lack of awareness may prevent access. To address this, the Australian Government funded Mackenzie's Mission­the Australian Reproductive Genetic Carrier Screening Project. Mackenzie's Mission aims to assess the acceptability and feasibility of an easily accessible RGCS program, provided free of charge to the participant. In study Phase 1, implementation needs were mapped, and key study elements were developed. In Phase 2, RGCS is being offered by healthcare providers educated by the study team. Reproductive couples who provide consent are screened for over 1200 genes associated with >750 serious, childhood-onset genetic conditions. Those with an increased chance result are provided comprehensive genetic counseling support. Reproductive couples, recruiting healthcare providers, and study team members are also invited to complete surveys and/or interviews. In Phase 3, a mixed-methods analysis will be undertaken to assess the program outcomes, psychosocial implications and implementation considerations alongside an ongoing bioethical analysis and a health economic evaluation. Findings will inform the implementation of an ethically robust RGCS program.

Paternal retraction of a fragile X allele to normal size, showing normal function over two generations.

The FMR1 premutation (PM:55-199 CGG) is associated with fragile X-associated tremor/ataxia syndrome (FXTAS) and when maternally transmitted is at risk of expansion to a hypermethylated full mutation (FM: ≥ 200 CGG) that causes fragile X syndrome (FXS). We describe a maternally transmitted PM (77 CGG) that was passed to a son (103 CGG), and to a daughter (220-1822 CGG), who were affected with FXTAS and FXS, respectively. The male with the PM showed low-level mosaicism for normal size of 30 and 37 CGG. This male had two offspring: one female mosaic for PM and FM (56, 157, >200 CGG) and another with only a 37 CGG allele detected in multiple tissues, neither with a clinical phenotype. The female with the 37 CGG allele showed normal levels of FMR1 methylation and mRNA and passed this 37 CGG allele to one of her daughters, who was also unaffected. These findings show that post-zygotic paternal retraction can lead to low-level mosaicism for normal size alleles, with these normal alleles being functional when passed over two generations.

FMR1 allele size distribution in 35,000 males and females: a comparison of developmental delay and general population cohorts.

PURPOSE: Developmental delay phenotypes have been associated with FMR1 premutation (PM: 55-200 CGG repeats) and "gray zone" (GZ: 45-54 CGG repeats) alleles. However, these associations have not been confirmed by larger studies to be useful in pediatric diagnostic or screening settings. METHODS: This study determined the prevalence of PM and GZ alleles in two independent cohorts of 19,076 pediatric referrals to developmental delay diagnostic testing through Victorian Clinical Genetics Service (cohort 1: N = 10,235; cohort 2: N = 8841), compared with two independent general population cohorts (newborn screening N = 1997; carrier screening by the Victorian Clinical Genetics Service prepair program N = 14,249). RESULTS: PM and GZ prevalence rates were not significantly increased (p > 0.05) in either developmental delay cohort (male PM: 0.12-0.22%; female PM: 0.26-0.33%; male GZ: 0.68-0.69%; female GZ: 1.59-2.13-%) compared with general population cohorts (male PM: 0.20%; female PM: 0.27-0.82%; male GZ: 0.79%; female GZ: 1.43-2.51%). Furthermore, CGG size distributions were comparable across datasets, with each having a modal value of 29 or 30 and ~1/3 females and ~1/5 males having at least one allele with ≤26 CGG repeats. CONCLUSION: These data do not support the causative link between PM and GZ expansions and developmental-delay phenotypes in pediatric settings.

Fracture during intravenous bisphosphonate treatment in a child with osteogenesis imperfecta: an argument for a more frequent, low-dose treatment regimen.

BACKGROUND/AIMS: Intravenous bisphosphonate therapy is the mainstay of medical treatment in osteogenesis imperfecta (OI) and has been shown to increase bone mass, decrease bone pain, improve mobility, and reduce the incidence of fractures. Sclerotic metaphyseal lines parallel to the growth plate are seen on long bone radiographs following cyclical intravenous therapy. These areas create stress risers within the bone that may act as foci for subsequent fractures as exemplified in this clinical case. METHODS: An 8-year-old girl with OI sustained a distal radial fracture following 3 years of treatment with 6-monthly intravenous zoledronate. Her diagnosis, response to treatment, and subsequent fracture at a sclerotic metaphyseal line is described. RESULTS: Peripheral quantitative computer tomography was used to characterise the presence of multiple stress risers at the distal forearm. Trabecular bone mineral density fluctuated from 34 to 126% compared to neighbouring 2-mm regions. CONCLUSION: There remain many unanswered questions about optimal bisphosphonate treatment regimens in children with OI. The formation of stress risers following intravenous bisphosphonate treatment raises the hypothesis that a more frequent and low-dose bisphosphonate regimen would provide more uniform dosing of bone in the growing child and reduce the likelihood of fractures compared to current treatment practices.

A case of periodic fever and persistent splenomegaly in a 2-year-old boy.

We report an unusual case of periodic fever associated with persistent splenomegaly in a 2-year-old boy. Febrile episodes occurred at regular 2 weekly intervals and each lasted for 1-2 days. Following resolution of the fever on each occasion, the patient developed neck stiffness of 1-2 days duration. The periodic febrile episodes, as well as the splenomegaly, spontaneously resolved 9 months after the onset of disease. Infectious and malignant causes were ruled out as far as possible. This patient's clinical features are unusual and do not match any known category of periodic fevers in childhood. We believe this case highlights the diagnostic challenges periodic fevers commonly represent.

An investigation of NOS2A promoter polymorphisms in Australian multiple sclerosis patients.

As with other major autoimmune diseases, susceptibility to multiple sclerosis (MS) is believed to result from the complex interaction of a number of genes, each with modest effect. Extensive research of experimental autoimmune encephalomyelitis in mice and several direct MS studies have implicated NOS2A, which encodes the inducible form of nitric oxide synthase, and the genetic region encoding NOS2A, 17q11.2, has been identified in a number of genome wide screens as being potentially associated with MS. We investigated four single nucleotide polymorphisms in the proximal promoter region of NOS2A, in a case-control group of 100 Australian MS patients and 100 controls and in 203 MS patients and their unaffected parents. We found a trend toward excess transmission of the -277A allele (tag for the AGCC haplotype) to HLA-DRB1*1501-positive MS patients (P (uncorrected)=0.05). We initially discovered a trend toward over-representation of the AGCC haplotype in HLA-DRB1*1501-positive compared to HLA-DRB1*1501-negative MS patients in the case-control cohort. However, when combined with the probands from the transmission disequilibrium analysis, this trend was nullified. Nonetheless, despite the lack of significant evidence of association for the NOS2A promoter polymorphisms with MS, the gene remains an interesting candidate for MS susceptibility, particularly with regard to the HLA-DRB1*1501 haplotype.

Analysis of the CCL3-L1 gene for association with HIV-1 susceptibility and disease progression.

CCL-L1 binds more potently to CCR5 than any other chemokine; it inhibits HIV-1 infection in vitro, and its gene occurs in variable copy numbers, some individuals failing to produce it. We analysed the frequency of the absence of CCL3-L1 in 268 Caucasian Australian HIV-1 patients and 260 uninfected individuals. A CCL-L1-negative genotype frequency of 2.3% was found in HIV-1 negative individuals. No association was found between the absence of CCL3-L1 and susceptibility to, or rate of progression of, HIV-1 infection.

Association of common T cell activation gene polymorphisms with multiple sclerosis in Australian patients.

Susceptibility to multiple sclerosis (MS) may be influenced by the interaction of several genes within a biological pathway. T cell activation and costimulation may be potentially important in MS pathogenesis. We have therefore investigated associations between MS and polymorphisms in the CD152 (CTLA-4), CD28, CD80 and CD86 genes in Australian patients. We found no significant MS association with CTLA-4 exon 1 +49 alleles, and meta-analysis showed no significant association across nine comparable datasets (OR=1.04, p=0.54), nor with primary progressive MS across seven datasets (OR=1.19, p=0.21). Haplotype analysis showed a trend towards a decrease of the CTLA-4-1722C, -1577G, +49G haplotype in +49 G positive MS patients compared with controls (p=0.06). Screening of CD28, CD80 and CD86 genes identified novel polymorphisms in the putative promoter regions of CD28 (-372 G/A) and CD86 (exon 2 -359 deletionAAG). There was a significant increase of the CD28 -372 G allele frequency in MS patients vs. controls (p=0.045) and a trend towards a significant interaction between this allele and the CTLA-4 +49 G allele (OR=4.00, p=0.058). Our results suggest that the CTLA-4 +49 alone is not associated with overall susceptibility to MS, but may be important in clinical subsets of patients and/or may interact epistatically with other gene polymorphisms.

Gene-gene interaction between the monoamine oxidase A gene and solute carrier family 6 (neurotransmitter transporter, noradrenalin) member 2 gene in anorexia nervosa (restrictive subtype).

We earlier found an association between anorexia nervosa (AN) restrictive subtype (AN-R) and an inserted sequence within the NETpPR, a polymorphic region located in the promoter of the solute carrier family 6 (neurotransmitter transporter, noradrenalin) member 2 (SLC6A2) gene. To further examine the noradrenergic system in AN-R we performed an association study with a functional polymorphism (MAOA-uVNTR) in the promoter of the monoamine oxidase A (MAOA) gene. Since monoamine oxidase A metabolises noradrenalin, a positive association with the MAOA gene would be biologically plausible. The transmission disequilibrium test and 95 trios/duos (AN-R females+biological parents) showed the main effect of the longer, more transcriptionally active form of the MAOA-uVNTR (MAOA-L) to be statistically non-significant (McNemar's chi(2)=1.4, df=1, P=0.238, odds ratio: 1.4, 95% CI 0.8-2.7). A case-control approach supported this finding. We then stratified the MAOA-uVNTR TDT data according to the (a) NETpPR genotype of the AN-R females, and (b) NETpPR allele transmitted from NETpPR-S4/L4 heterozygous mothers. In both cases, contingency table analysis revealed previously unreported gene-gene interaction between the MAOA and SLC6A2 genes (P=0.019 and 0.019, respectively). Receiving an MAOA-L allele more than doubles the risk for developing AN-R, conditional on an individual also being a NETpPR-L4 homozygote (stratum-specific odds ratio: 2.4, 95% CI 1.1-6.0). These results suggest important involvement of the noradrenergic system in the biological underpinnings of AN-R.

Investigation of epistasis between the serotonin transporter and norepinephrine transporter genes in anorexia nervosa.

Weight-restored patients with anorexia nervosa (AN) respond favorably to the selective serotonin reuptake inhibitor fluoxetine, which justifies association studies of the serotonin transporter gene (SLC6A4, alias SERT) and AN. Case-control studies suggest that the least transcriptionally active allele of the SERT gene promoter polymorphism (5-HTTLPR) has an increased frequency in AN patients. However, this finding was not replicated with 55 trios (AN child+parents) and the transmission disequilibrium test (TDT). To clarify the role of the 5-HTTLPR in susceptibility to AN, we used the TDT and 106 Australian trios to provide 93% power to detect a genotypic relative risk (GRR) of 2.0. Our results were negative for this GRR (McNemar's chi(2)=0.01, df=1, p=0.921, odds ratio 1.0, 95% CI 0.7-1.5). Additionally, we found no association with AN females, AN subtype, age at onset, or minimum BMI. We then performed the first reported investigation of epistasis between the SERT gene and norepinephrine transporter gene (SLC6A2, alias NET) in AN, as an earlier study suggested that atypical AN responds to the dual serotonin-norepinephrine reuptake inhibitor venlafaxine. We observed no epistasis between the 5-HTTLPR and a polymorphism within the NET gene promoter polymorphic region (NETpPR) (chi(2)=0.48, df=1, p=0.490). Although 5-HTTLPR modulates serotonin reuptake by the serotonin transporter, our analyses provide no evidence that susceptibility to AN is modified by 5-HTTLPR alone, nor in concert with as yet undetermined functional effects of the NETpPR polymorphism.

Identification of 11 novel and common single nucleotide polymorphisms in the interleukin-7 receptor-alpha gene and their associations with multiple sclerosis.

We have investigated the interleukin-7 receptor (IL-7R) alpha-chain gene as a positional and functional candidate gene for susceptibility to multiple sclerosis (MS), in view of its chromosomal location on 5p14-p12, a region that has shown suggestive linkage in MS genome screens, and its role in T- and B-cell proliferation and reactivity. Amplification and DNA sequencing of the IL-7Ralpha gene in pooled and individual samples identified 13 single nucleotide polymorphisms (SNPs), 11 of which are novel, including three in the promoter region, three in exons encoding amino-acid changes (ACC(Thr)66ATC(Ile), ATC(Ile)244ACC(Thr), ATC(Ile)336GTC(Val)), four in introns and one in the 3' untranslated region. Four IL-7R haplotypes were identified for nine SNPs, showing linkage disequilibrium across the gene, and allowing haplotype frequency determination from just three of the nine SNPs. Genotyping of the -504 polymorphism in 101 MS and 90 controls showed a suggestive (P=0.1) association of the T allele with MS; however, this was not supported by transmission disequilibrium testing in 186 MS trio families (P=0.8). There were trends towards an increase of the GTG+ haplotype (odds ratio=1.45), and under-representation of the TTA+ haplotype (OR=0.65) in DRB1*1501-positive MS cases, suggesting that larger sample sizes and comparison in more defined MS patient groups may support an association with the IL-7R gene. These polymorphisms would also be useful for studying genetic associations with other immunologic diseases.