X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

Weakly acidic pH reduces inflammatory cytokine expression in airway epithelial cells.

BACKGROUND: Aspiration lung disease (ALD) is a common cause of respiratory morbidity in children and adults with severe neurodisability (sND). Recent studies suggest that chronic microaspiration of gastric contents is associated with mild rather than low, airway acidification. We investigated inflammatory responses to infection by airway epithelial cells (AECs) exposed to weakly acidic media. METHODS: Using pH measurements from children with sND at high risk of ALD as a guide, we incubated AECs in weakly acidic (pH5.5-7.4) media alone; in combination with lipopolysaccharide (LPS); or prior to LPS stimulation at normal pH. Interleukin (IL) -6 and IL-8 expression were measured. RESULTS: IL-6/8 expression in AECs simultaneously exposed to weakly acidic media and LPS for 4 h was reduced with no effect on cell viability. Pre-incubation of AECs at weakly acidic pH also reduced subsequent LPS-induced cytokine expression. Suppression of inflammation was greatest at lower pHs (pH 5.5-6.0) for prolonged periods (16/24 h), but this also adversely affected cell viability. CONCLUSION: AEC inflammatory responses to bacterial stimuli is markedly reduced in a mildly acidic environment.

Ten-year changes in positive and negative marker food, fruit, vegetables, and salad intake in 9-10 year olds: SportsLinx 2000-2001 to 2010-2011.

BACKGROUND: To investigate changes in intakes of 'negative' and 'positive' foods, fruit, vegetables, and salad in serial cohorts of 9-10-year-old children from 2000-2001 to 2010-2011. METHODS: For this serial, cross-sectional study, children in school year 5 (9-10 years of age) completed the SportsLinx Lifestyles Survey [n = 30,239 (15,336 boys and 14,903 girls)]. Changes in positive and negative food scores, and the proportion of boys and girls reportedly consuming fruit, vegetables and salad on the previous day to surveying, were investigated annually from 2000 to 2011. RESULTS: The consumption of negative foods declined and positive foods increased significantly compared to baseline. Positive changes in fruit, vegetables and salad consumption were observed over time, with the most recent cohort more likely to consume fruit, vegetables and salad compared to the 2000-2001 baseline. Girls displayed more favourable positive and negative food scores and were more likely to consume fruit, salad and vegetables across several study years compared to boys. CONCLUSIONS: The consumption of negative and positive foods, fruit, vegetables, and salad has improved over the last 10 years. In addition, girls appear to have better positive and negative food scores, and were more likely to consume fruit, vegetables and salad, across a number of study years or cohorts compared to boys. These encouraging findings suggest that children's food intake has improved since 2000. Furthermore, the data indicate that boys and girls may require separate or different healthy eating messages to further improve food intake.

Genotype and clinical care correlations in craniosynostosis: findings from a cohort of 630 Australian and New Zealand patients.

Craniosynostosis is one of the most common craniofacial disorders encountered in clinical genetics practice, with an overall incidence of 1 in 2,500. Between 30% and 70% of syndromic craniosynostoses are caused by mutations in hotspots in the fibroblast growth factor receptor (FGFR) genes or in the TWIST1 gene with the difference in detection rates likely to be related to different study populations within craniofacial centers. Here we present results from molecular testing of an Australia and New Zealand cohort of 630 individuals with a diagnosis of craniosynostosis. Data were obtained by Sanger sequencing of FGFR1, FGFR2, and FGFR3 hotspot exons and the TWIST1 gene, as well as copy number detection of TWIST1. Of the 630 probands, there were 231 who had one of 80 distinct mutations (36%). Among the 80 mutations, 17 novel sequence variants were detected in three of the four genes screened. In addition to the proband cohort there were 96 individuals who underwent predictive or prenatal testing as part of family studies. Dysmorphic features consistent with the known FGFR1-3/TWIST1-associated syndromes were predictive for mutation detection. We also show a statistically significant association between splice site mutations in FGFR2 and a clinical diagnosis of Pfeiffer syndrome, more severe clinical phenotypes associated with FGFR2 exon 10 versus exon 8 mutations, and more frequent surgical procedures in the presence of a pathogenic mutation. Targeting gene hot spot areas for mutation analysis is a useful strategy to maximize the success of molecular diagnosis for individuals with craniosynostosis.

Transcriptome profiling of UPF3B/NMD-deficient lymphoblastoid cells from patients with various forms of intellectual disability.

The nonsense-mediated mRNA decay (NMD) pathway was originally discovered by virtue of its ability to rapidly degrade aberrant mRNAs with premature termination codons. More recently, it was shown that NMD also directly regulates subsets of normal transcripts, suggesting that NMD has roles in normal biological processes. Indeed, several NMD factors have been shown to regulate neurological events (for example, neurogenesis and synaptic plasticity) in numerous vertebrate species. In man, mutations in the NMD factor gene UPF3B, which disrupts a branch of the NMD pathway, cause various forms of intellectual disability (ID). Using Epstein Barr virus-immortalized B cells, also known as lymphoblastoid cell lines (LCLs), from ID patients that have loss-of-function mutations in UPF3B, we investigated the genome-wide consequences of compromised NMD and the role of NMD in neuronal development and function. We found that ~5% of the human transcriptome is impacted in UPF3B patients. The UPF3B paralog, UPF3A, is stabilized in all UPF3B patients, and partially compensates for the loss of UPF3B function. Interestingly, UPF3A protein, but not mRNA, was stabilised in a quantitative manner that inversely correlated with the severity of patients' phenotype. This suggested that the ability to stabilize the UPF3A protein is a crucial modifier of the neurological symptoms due to loss of UPF3B. We also identified ARHGAP24, which encodes a GTPase-activating protein, as a canonical target of NMD, and we provide evidence that deregulation of this gene inhibits axon and dendrite outgrowth and branching. Our results demonstrate that the UPF3B-dependent NMD pathway is a major regulator of the transcriptome and that its targets have important roles in neuronal cells.

Nfasc155H and MAG are specifically susceptible to detergent extraction in the absence of the myelin sphingolipid sulfatide.

Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

Screening and cell-based assessment of mutations in the Aristaless-related homeobox (ARX) gene.

ARX mutations cause a diverse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA samples from 613 individuals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred samples without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp); and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.

Changes in fitness, body mass index and obesity in 9-10 year olds.

BACKGROUND: The prevalence of obesity in children has increased substantially in recent years and, paediatric obesity and poor fitness are risk factors for disease. The present study aimed to assess changes in body mass index (BMI), the prevalence of obesity and changes in aerobic endurance over time in 9-10-year-old schoolchildren. METHODS: Participants were recruited by the SportsLinx project from primary schools across Liverpool. Height and weight data were used to calculate BMI. The prevalence of obesity and overweight were estimated using age- and sex-specific cut-off points. Performance on the 20-m multi-stage shuttle runs test (20 mMST) was used as a marker of aerobic endurance. Data were available for 13,418 (6572 boys, 6846 girls) 9-10-year-old children. Analysis of covariance was completed to assess year-on-year changes in BMI controlling for deprivation (IMD) and 20 mMST performance, and 20 mMST performance controlling for IMD and BMI. RESULTS: No significant changes in BMI from baseline were observed (P > 0.05). Obesity prevalence reduced in girls (2005 = 10.3%, 2008 = 8.52% in 2008). The data for boys showed no reductions in prevalence (2005 = 6.77%, 2008 = 7.87%). The most recent cohort for boys and two most recent cohorts for girls had lower levels of aerobic endurance than baseline (2004-2005) (P

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Coping mechanisms used by women in an attempt to avoid symptoms of chronic radiation enteritis.

BACKGROUND: Approximately 12,000 individuals undergo pelvic radiotherapy in the UK every year and up to 50% may develop symptoms of chronic radiation enteritis (CRE). Health professionals often give inappropriate dietary advice to patients in an attempt to avoid CRE symptoms, such as avoiding fibre, despite a lack of evidence to support this. METHODS: The present study aimed to explore dietary advice and changes made to the diet by women treated with pelvic radiotherapy. A questionnaire was distributed to 117 women attending Liverpool Women's Hospital, exploring symptoms of CRE and asking questions about diet and medication advice received, as well as changes made to the diet. RESULTS: Ninety-five (87.2%) women completed the questionnaire and 47% had changed their diet. No significant relationship was observed between receiving dietary advice and making changes to the diet (P > 0.05), although those advised by a dietitian were more likely to be taking regular anti-diarrhoeal medication (P < 0.05) and those taking regular medication found that it helped most/all of the time (P < 0.001). CONCLUSIONS: CRE sufferers should be dissuaded from unnecessarily restricting their diet, which may result in malnutrition. Regular follow-up screening should lead to the identification of sufferers and the offer of individual advice if necessary.

Identification of 'hot spots' of obesity and being underweight in early pregnancy in Liverpool.

BACKGROUND: Obesity and being underweight in pregnancy are related to an increased risk of maternal and foetal morbidity, yet their prevalence is often unknown. The present study aimed to identify neighbourhoods with a higher than average prevalence or 'hot spots' of obesity and/or being underweight among first trimester pregnant women. METHODS: A database was compiled consisting of postcode, height and weight for 7981 women who had booked-in for antenatal care between July 2004 and June 2005 at Liverpool Women's Hospital. Body mass index (BMI) was calculated and women were categorised accordingly. Postcodes for 6865 cases across Merseyside were converted to geolocations (pin-points on a map) using conversion software (http://www.census.ac.uk/cdu/). RESULTS: There was a very high prevalence of being overweight (27%) and obesity (17%); 3.8% of women were underweight and probably malnourished (BMI < 18.5 kg m(-2)); and a further 10.7% of women were possibly malnourished (BMI < 20.0 kg m(-2). Deriving case density from the geolocations allowed visualisation and identification of six neighbourhoods with above average levels of obesity and three neighbourhoods had marked concentrations of both being underweight and obesity. CONCLUSIONS: These neighbourhoods, particularly those identified as 'hot spots' for both being underweight and obesity, include some of the most deprived wards in the UK. As dietetic intervention may help to promote optimal weight gain during pregnancy and improve dietary intake for pregnant women and their families, primary health care providers should target these localities with a high prevalence of low and high BMI as a priority.

An evaluation of an attempt to change the snacking habits of pre-school children using social marketing.

OBJECTIVES: To demonstrate the feasibility and value of social marketing over a wide geographical footprint, and to improve the snacking habits of pre-school children. STUDY DESIGN: Two cross-sectional studies before and approximately 3 months after a social marketing intervention targeted into the least affluent areas using convenience sampling. METHODS: Based on market research and directed at the least affluent areas, a brand was created ('Snack Right') and an information leaflet was distributed, supported by a media launch and events at children's centres in targeted areas. This evaluation of some aspects of the project is based on a questionnaire delivered before and after the events. RESULTS: There were several differences in pre- and post-event responses consistent with the messages delivered, for example increased spending on fruit (but not vegetables) and more positive attitudes towards fruits and vegetables. Some ambiguities were exposed, for example towards snacking, which have implications for the nutritional knowledge of health professionals. The Snack Right brand was recalled by a very high proportion of respondents at follow-up. CONCLUSIONS: This project has shown that social marketing is a viable tool at a subregional level and has the potential to change attitudes, knowledge and behaviour.

n of 1 trial for an ornithine transcarbamylase deficiency carrier.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder of the urea cycle. It is often fatal in affected males. Treatment for affected individuals includes dietary protein restriction, activation of alternative pathways of nitrogen excretion and L-arginine supplementation. Depending on the amount of X chromosome inactivation skewing, females show variable clinical manifestations, and sometimes the need for treatment, including medications, is unclear. We conducted an n of 1 randomized controlled trial on an obligate OTC carrier. The treating physician and patient were blinded to treatment. Either placebo capsules or L-arginine capsules were given for weekly periods. Weekly efficacy indicators included plasma arginine and glutamine levels and a quality of life/mood assessment questionnaire scale. Clear evidence of benefit with L-arginine compared to placebo was shown. This is the first time an n of 1 randomized controlled trial has been reported for an X-linked metabolic condition. Despite some logistic hurdles, we have demonstrated that this method was an effective tool for determining the value of treatment. We propose that other rare metabolic conditions may be amenable to such trials, if the benefit of treatment is in doubt.

Mapping dietary habits may provide clues about the factors that determine food choice.

BACKGROUND: Food deserts are thought to be a barrier to making healthier food choices. This concept has been challenged. The interaction between the physical environment and children's food choice has received little attention. The present study used food intake data to generate hypotheses concerning the role of the physical environment in food choice. METHODS: A cross-sectional analysis was conducted of the dietary habits of Year 5 (9-10-year-old) children from 90 of Liverpool's 118 primary schools. Individuals with the 'best' and 'worst' food choices were mapped and two areas associated with these extreme choices located. RESULTS: One thousand five hundred and thirty-five children completed the dietary questionnaire and supplied a full and valid postcode. Two adjacent areas with relatively large numbers of children in the 'best' and 'worst' food choice groups were chosen. Both areas had very similar socio-economic profiles. The contrast in the physical environments was striking, even on visual inspection. CONCLUSIONS: Food deserts as a cause of poor food choice did not stand scrutiny; the area located by the worst food choices had a plethora of shops selling food (better termed a food prairie), whereas the area located by the best food choices had no shops in evidence but did have more 'space'.

Tumor-specific cell-mediated immunity in household contacts of cancer patients..

Patients with osteogenic sarcoma (and related tumors), hypernephroma, and breast carcinoma, and their household contacts were tested for tumor-specific cell-mediated immunity against these tumors with the use of a short-term chromium-51 release assay. This assay, reproducible over many months and well-correlated with the clinical course of the patients, was used to demonstrate that household contacts of patients with osteogenic sarcoma and breast carcinoma have specific immunity against the tumor type with which they have been in contact. In both types of tumors, the range of cytotoxicity values produced by lymphocytes from the household contacts was significantly higher than that of the normal population. The incidence of immunity was much higher in household contacts of patients with breast carcinoma than in those of patients with osteogenic sarcoma. Immunity was found with equal frequency in men and women, as well as in genetically and nongenetically related household contacts (guardians, adopted children, spouses). Immunity against hypernephroma was not demonstrated in either patients with hypernephroma or their household contacts.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

NF-kappaB p65 (RelA) homodimer uses distinct mechanisms to recognize DNA targets.

BACKGROUND: The NF-kappaB family of dimeric transcription factors regulates the expression of several genes by binding to a variety of related DNA sequences. One of these dimers, p65(RelA), regulates a subclass of these targets. We have shown previously that p65 binds to the 5'-GGAA T TTTC-3' sequence asymmetrically. In that complex one subunit base specifically interacts with the preferred 5' half site and the other subunit binds non-specifically to the 3' half site. RESULTS: Here we describe the crystal structures of two new p65-DNA complexes. One complex contains a pseudosymmetric 5'-GGAA T TTCC-3' DNA sequence taken from the enhancer of the gene encoding interleukin 8 (IL-8) and the other contains the asymmetric 5'-GGAA T TCCC-3' target DNA taken from the enhancer of the gene encoding type VII collagen. As expected, the global positioning of the dimer on both DNA targets is roughly symmetric, however, the hydrogen-bonding patterns at the protein-DNA interfaces differ significantly. One of the p65 monomers in complex with the asymmetric DNA binds to an extra base pair located immediately upstream of the 5'-GGAA-3' half site. We also show that p65 binds to these targets with almost equal affinity and that different residues have variable roles in binding different kappaB targets. CONCLUSIONS: Taken together, these structures reveal that p65 exhibits the unique capability to specifically bind DNA targets of variable lengths from four to ten base pairs. Also, the small protein segment Arg41-Ser42-Ala43 is at least partially responsible for flexibility in DNA-binding modes.

Internal antigens accessible in breast cancer: implications for tumor targeting.

Proponents of monoclonal antibody (MAb)-mediated cancer therapy often assume that a major limitation in clinical application of MAbs is their lack of absolute specificity for malignant cells. In addition, the presence of surface target antigens is thought to be essential. These requirements may be more stringent than necessary for the clinical usefulness of MAbs. We have demonstrated selective localization of a MAb to keratin polypeptides in malignant breast epithelium under conditions of passive infusion of antibody in fresh surgical specimens of breast carcinoma. Although these proteins are normal intracellular constituents of epithelial cells throughout the body, localization of antikeratin antibodies only within the tumor population is most probably associated with the presence of cells permeable to macromolecules. This permeable tumor cell fraction could be recruited for targeting neighboring impermeable tumor cells with radioisotopes or other antitumor agents conjugated to antibodies directed against intracellular antigens.

Expression of basal and luminal epithelium-specific keratins in normal, benign, and malignant breast tissue.

We characterized the subclass-specific keratins in the epithelium of normal, benign, and malignant breast tissue. Monoclonal antibody 34BE12 stained luminal as well as basal epithelium in normal and benign specimens and all tumor cells in malignant specimens. Antibody 312CS-1 reacted only with basal cells, and antibody LE61 reacted only with luminal cells in the normal and benign specimens. In 34 of 36 breast carcinomas examined, the basal and luminal cell-specific antibodies showed complementary patterns of reactivity, while in the remaining 2 specimens, neither antibody was reactive. The findings reported in this study demonstrate that expression of subclass-specific keratins is mutually exclusive not only in normal and benign mammary specimens but also in breast carcinoma. These findings suggest a role for epithelial subclass-specific antibodies in the histogenetic and prognostic subclassifications of breast carcinoma.