Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility

Lymph node homing cells biologically enriched for γδ T cells express multiple genes from the T19 repertoire

Sheep γδ T cells have been shown serologically to express T19, a membrane protein of 180-200 kDa which is a member of the scavenger receptor superfamily. Previous work from this laboratory resulted in the detection of a multigene family of T19-like genes in the sheep genome. In this study nucleotide sequences from several T19 genes were determined and are reported along with the corresponding segments of a number of expressed mRNA molecules. A segment of a single sheep T19-like gene was sequenced and these data, along with the corresponding sequences from cloned T19-like cDNA molecules from sheep and cow, were used to design an ollgonucleotide primer system suitable for amplification of corresponding segments of many T19 genes and their cDNAs. Between 30 and 40% of cloned T19 genes were amenable to amplification using the selected primers, and sequence analysis of cloned PCR products confirmed that different T19 genes encode unique amino acid sequences. The expression of multiple T19 genes was established using cDNA molecules obtained from a single sample of sheep lymphocyte mRNA. The possible role of the T19 family of genes is discusse

Impact of soil warming and shading on colonization and community structure of arbuscular mycorrhizal fungi in roots of a native grassland

Arbuscular mycorrhizal (AM) fungi have a major influence on the structure, responses and below-ground C allocation of plant communities. Our lack of understanding of the response of AM fungi to factors such as light and temperature is an obstacle to accurate prediction of the impact of global climate change on ecosystem functioning. In order to investigate this response, we divided a grassland site into 24 plots, each either unshaded or partly shaded with soil either unheated or heated by 3°C at 2 cm depth. In both short-term studies in spring and autumn, and in a 1-year-long study, we measured root length colonization (LRC) by AM and non-AM fungi. For selected root samples, DNA sequences were amplified by PCR with fungal-specific primers for part of the small sub-unit (SSU) rRNA gene. In spring, the total LRC increased over 6 weeks from 12% to 25%. Shading significantly reduced AM but increased non-AM fungal colonization, while soil warming had no effect. In the year-long study, colonization by AM fungi peaked in summer, whereas non-AM colonization peaked in autumn, when there was an additive effect of shading and soil warming that reduced AM but increased non-AM fungi. Stepwise regression revealed that light received within the 7 days prior to sampling was the most significant factor in determining AM LRC and that mean temperature was the most important influence on non-AM LRC. Loglinear analysis confirmed that there were no seasonal or treatment effects on the host plant community. Ten AM fungal sequence types were identified that clustered into two families of the Glomales, Glomaceae and Gigasporaceae. Three other sequence types were of non-AM fungi, all Ascomycotina. AM sequence types showed seasonal variation and shading impacts: loglinear regression analysis revealed changes in the AM fungal community with time, and a reduction of one Glomus sp. under shade, which corresponded to a decrease in the abundance of Trifolium repens. We suggest that further research investigating any impacts of climate change on ecosystem functioning must not only incorporate their natural AM fungal communities but should also focus on niche separation and community dynamics of AM fungi

Responsible implementation of expanded carrier screening.

This document of the European Society of Human Genetics contains recommendations regarding responsible implementation of expanded carrier screening. Carrier screening is defined here as the detection of carrier status of recessive diseases in couples or persons who do not have an a priori increased risk of being a carrier based on their or their partners' personal or family history. Expanded carrier screening offers carrier screening for multiple autosomal and X-linked recessive disorders, facilitated by new genetic testing technologies, and allows testing of individuals regardless of ancestry or geographic origin. Carrier screening aims to identify couples who have an increased risk of having an affected child in order to facilitate informed reproductive decision making. In previous decades, carrier screening was typically performed for one or few relatively common recessive disorders associated with significant morbidity, reduced life-expectancy and often because of a considerable higher carrier frequency in a specific population for certain diseases. New genetic testing technologies enable the expansion of screening to multiple conditions, genes or sequence variants. Expanded carrier screening panels that have been introduced to date have been advertised and offered to health care professionals and the public on a commercial basis. This document discusses the challenges that expanded carrier screening might pose in the context of the lessons learnt from decades of population-based carrier screening and in the context of existing screening criteria. It aims to contribute to the public and professional discussion and to arrive at better clinical and laboratory practice guidelines.European Journal of Human Genetics advance online publication, 16 March 2016; doi:10.1038/ejhg.2015.271

The expectations and realities of nutrigenomic testing in australia: A qualitative study.

BACKGROUND: Consumer genomic testing for nutrition and wellness, (nutritional genomics), is becoming increasingly popular. Concurrently, health-care practitioners (HPs) working in private practice (including doctors interested in integrative medicine, private genetic counsellors, pharmacists, dieticians, naturopaths and nutritionists) are involved as test facilitators or interpreters. OBJECTIVE: To explore Australian consumers' and HPs' experiences with nutrigenomic testing. METHOD: Semi-structured in-depth interviews were conducted using predominantly purposive sampling. The two data sets were analysed individually, then combined, using a constant comparative, thematic approach. RESULTS: Overall, 45 interviews were conducted with consumers (n = 18) and HPs (n = 27). Many of the consumer interviewees experienced chronic ill-health. Nutrigenomic testing was perceived as empowering and a source of hope for answers. While most made changes to their diet/supplements post-test, self-reported health improvements were small. A positive relationship with their HP appeared to minimize disappointment. HPs' adoption and views of nutrigenomic testing varied. Those enthusiastic about testing saw the possibilities it could offer. However, many felt nutrigenomic testing was not the only 'tool' to utilize when offering health care. DISCUSSION: This research highlights the important role HPs play in consumers' experiences of nutrigenomics. The varied practice suggests relevant HPs require upskilling in this area to at least support their patients/clients, even if nutrigenomic testing is not part of their practice. PATIENT OR PUBLIC CONTRIBUTION: Advisory group included patient/public group representatives who informed study design; focus group participants gave feedback on the survey from which consumer interviewees were sourced. This informed the HP data set design. Interviewees from HP data set assisted with snowball sampling

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Metabolism and Cytotoxicity of Aflatoxin B1 in Rat Liver Cells.

Freshly isolated rat hepatocytes were found to metabolise 14C-aflatoxin B1 (AFB1) to aflatoxins M1 (AFM1) and Q1(AFQ1), and to radiolabelled polar material, presumably conjugates, as analysed by high-performance liquid chromatography, thin-layer chromatography and radioactive counting. Feeding AFB1 to rats enhanced production of polar material only, whereas phenobarbitone and 3-methylcholanthrene (3-MC) pretreatments both enhanced the rate of AFB1 metabolism and the production of polar material, with increased AFQ1 and AFM1 respectively. Formation of AFQ1 appeared to be mediated via cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes appeared to be involved in AFM1 production. Hepatocytes cultured for 24 hr from control and pretreated rats also metabolised AFB1 but not as efficiently as the corresponding freshly isolated cells. Metabolism of AFB1 and cytochrome P450 contents were undetected in a liver-derived epithelial-like dividing cell line, BL8L. AFB1 caused marked and rapid (1 hr) inhibition of ribonucleic acid (RNA) synthesis and pronounced but less rapid inhibition of protein synthesis. In both freshly isolated and 24 hr cultured hepatocytes from control rats (AFB1 concentration, 0.01-0.5 μq ml-1). Pretreatments with phenobarbitone and 3-MC resulted in reduced AFB1 inhibition of RNA and protein syntheses at the lower concentrations of AFB1 only, whereas cells from AFB1 fed rats were much less susceptible to AFB1 inhibition of these processes at all concentrations. In BL8L cells, AFB1 caused considerable inhibition of deoxyribonucleic acid (DNA) synthesis following 24 hr incubation, but inhibition of RNA and protein syntheses was minimal. In cells from control rats, AFB1 addition resulted in pronounced cytotoxicity (as assessed by morphological criteria of viability and trypan blue exclusion) following 4 hr incubation. However, in cells from phenobarbitone and 3-MC pretreated rats, cytotoxicity was marked at high AFB1 concentrations, whereas AFB1 feeding caused resistance to the cytotoxic action of AFB1 at all concentrations. The BL8L cells were much less susceptible to AFB1 cytotoxicity than in the hepatocytes. It is concluded that cytotoxicity of AFB1 is increased as a result of metabolism and that AFB1 feeding, in particular, confers resistance against AFB1 cytotoxicity

Integrating genetics as practices of primary care

This study examines the responses of general practitioners (GPs) in Vic., Australia to an increased emphasis on genetics in primary care. A qualitative analysis of focus group interviews with GPs in regional and metropolitan areas and one focus group interview with genetics experts showed that despite the emphasis placed on genetics by the experts, GPs remained ambivalent to the routine integration of genetics into general practice. This response from GPs has been noted in several studies and is most commonly attributed to GPs' lack of knowledge about genetics. In this study we argue that a 'cognitive deficit' understanding of the problem excludes many of the factors that GPs regard as important in relating genetics to primary care. We show that GPs' ambivalence emerges from how they situate genetics within practices of patient care and in relation to what they regard as good patient management. We found that GPs respond most enthusiastically to genetics and genetic testing if they feel it changes their management in ways they consider of benefit to their patients. GPs have specialist skills in managing the heterogeneity of patient care and these skills enable them to situate genetics relative to the overall needs of patients. Preparing GPs to 'do' genetics when the need arises by finding ways to make genetics information available to them as the need arises would facilitate the integration of genetics as practices of primary care.General practitioners Primary care Genetics education Australia