ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome

It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with alpha thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.      

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

Lack of induction of micronuclei in human peripheral blood lymphocytes treated with hydroquinone

Hydroquinone (HQ) has been reported to produce chromosomal effects in some in vivo and in vitro animal models. Its potential for inducing similar effects in human lymphocytes is less clear. The purpose of this study was to examine human lymphocytes treated with HQ for the presence of chromosomal anomalies, using an accepted assay for micronuclei. In addition, the stability of HQ in culture medium was determined to verify exposures. Lymphocyte cultures were obtained from eight donors so that variable responses amongst individuals could be assessed. The micronucleus assays utilized were a common 72 h assay with no wash, as well as two assay variations to maximize cell division. Assay variations consisted of either cell washing at 44 h or allowing unwashed cultures an extra 24 h recovery period before harvest. In all assays treatment was at 24 h post-mitogenic stimulation and cytochalasin B was added to stop dividing cells from undergoing cytokinesis. Thus, cells that were scored had undergone one division in the presence of the chemical. Stability results showed that while HQ was detectable in cultures at least for 15 h, it was considerably more stable at 25 than at 100 or 250 microM treatment levels. Results generated using any of the three micronucleus assay variations showed no significant increase in micronuclei in cultures treated with 12.5-200 microM HQ. Colchicine, the positive control and a known spindle disrupter, produced elevated levels of micronuclei. At certain HQ concentrations, a block in cell division was observed, as evidenced by a decrease in percent binucleated cells and replicative index end-points. By varying the assay conditions, cell cultures overcame this block in division and divided at HQ concentrations up to 200 microM, depending on the donor. The reversible block in cell division observed may be a protective response, allowing cells to recover without gross chromosomal damage. This study has substantially expanded the database with regard to the effects of HQ treatment on lymphocytes.     

Exploring the utility of whole‐exome sequencing as a diagnostic tool in a child with atypical episodic muscle weakness

The advent of whole‐exome next‐generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½‐year old female patient with a 2‐year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work‐up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di‐deoxy sequencing. WES revealed a de novo non‐synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.     

Serum concentrations of oestradiol-17beta, progesterone, relaxin and chorionic gonadotrophin during blastocyst implantation in natural pregnancy cycle and in embryo transfer cycle in the rhesus monkey

The present study was undertaken to assess the temporal association between the profiles of serum concentrations of oestradiol-17beta, progesterone, chorionic gonadotrophin (CG) and relaxin in pregnancies established naturally, and after embryo transfer, as well as in failed pregnancies in rhesus monkeys. In naturally mated cycles (group 1) a conception rate of 75% was obtained. In group 1, the mean day of CG detection in serum was 11.5 +/- 1.9 day post-ovulation, and for relaxin, 9.0 +/- 2.5 day post-ovulation. In group 2, embryo transfer to synchronous, non-mated surrogate recipients was performed; seven embryo transfer cycles yielded three pregnancies which were allowed to continue to term and normal infants were delivered. In embryo transfer cycles the mean day of CG detection was 14.8 +/- 1.8 day post- ovulation, and for relaxin, 11.4 +/- 2.6 day post-ovulation. A delay of about 3 days was observed in the appearance in circulation of CG (P < 0.05) and also of relaxin (P < 0.05) between natural mated and embryo transfer conception cycles. Significant differences (P < 0.05 for progesterone and P < 0.03 for oestradiol) were obtained for the areas under the curves for progesterone and oestradiol between days 12 and 16 in conception cycles compared with failed pregnancies. These data provide the first observation of the normal hormonal signals associated with maternal recognition of transferred embryos during the peri- implantation period, and suggest that the use of such an experimental primate embryo transfer model may help to elucidate components of maternal and embryonic signal-response mechanisms during embryo implantation.      

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub- domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non- homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.      

Enhanced functional response to CXCL12/SDF-1 through retroviral overexpression of CXCR4 on M07e cells: implications for hematopoietic stem cell transplantation

The chemokine CXCL12 (stromal cell derived factor-1/SDF-1) stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. CXCL12 is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. Previous studies suggest that breaking the CXCL12-CXCR4 interaction mobilizes HPCs, blocking CXCR4 inhibits HSC homing, and overexpression increases HSC/HPC repopulation. The efficiency of mobilization and engraftment therefore appears to be dependent on the response of HSCs/HPCs to CXCL12, which is in turn dependent upon levels of CXCR4 expressed on HSCs/HPCs. However, expression of CXCR4 on the surface of HSCs/HPCs appears to be variable. To study the function of CXCR4 on HSCs/HPCs, we used the MSCV-based bicistronic (EGFP) retroviral vector MIEG3 to overexpress CXCR4 on M07e cells, an established model of human HPC. CXCR4 overexpression resulted in significant increases in CXCL12-induced chemotaxis and cell survival. Most importantly, cells overexpressing CXCR4 responded to CXCL12 at levels typically too low induce a response. These data suggest that an increased transplant efficiency resulting from CXCR4 overexpression is likely a function of increased HSC/HPC homing and increased HSC/HPC survival in the recipient's BM. These experiments also validate the ability of the MIEG3-CXCR4 retroviral construct to overexpress CXCR4 efficiently and the use of MIEG3-CXCR4 M07e cells for further study. Finally, this information may have future potential therapeutic implications for improvements in transplant efficiency.     

Hormone therapy and breast cancer: what factors modify the association?

OBJECTIVE: To determine factors that may modify the association between hormone therapy (HT) and breast cancer risk. DESIGN: Prospective cohort study (the Melbourne Collaborative Cohort Study) of 24,479 women aged 40 to 69 years. History of HT use was collected at baseline and 4 years later by questionnaire. By June 2002, 336 cases of breast cancer were diagnosed among 13,444 women postmenopausal at baseline. Association of breast cancer risk with history of HT use was analyzed using proportional hazards models. RESULTS: The hazard ratio (HR) for recent HT use (current or stopped within the last year) was elevated (HR 1.51; 95% CI, 1.16-1.98) but was not significantly increased for past HT users (HR 1.19; 95% CI, 0.86-1.64). Recent HT use was associated with better differentiated tumors but was not more likely to be associated with estrogen receptor-positive / progesterone receptor-positive tumors. There was little evidence of interactions between recent HT use and body mass index, alcohol intake, parity, and smoking, although the HR for recent HT use in categories of alcohol consumption was greatest in women consuming the most alcohol (HR 2.37; 95% CI, 1.45-3.88 for those consuming > or = 10 g/d versus HR 1.33; 95% CI, 0.85-2.08 for nondrinkers, P interaction = 0.32). CONCLUSIONS: The risk of breast cancer for recent users of HT in this Australian population is increased by approximately 50%. Our results suggest that any potential modifying effect of the association between HT and breast cancer risk by factors such as alcohol intake and body mass index is likely to be modest.     

Child-parent attachment following early institutional deprivation

Child-parent attachment quality with an adoptive caregiver at age 4 years was examined in a sample of 111 children adopted into the United Kingdom following early severe deprivation in Romania and a comparison group of 52 nondeprived within-United Kingdom adoptees. Findings indicated that, compared with nondeprived adoptees, children who experienced early severe deprivation were less likely to be securely attached and more likely to show atypical patterns of attachment behavior; ordinary forms of insecure attachment were not associated with deprivation. Within the sample of deprived adoptees, there was a dose-response association between duration of deprivation and disturbances in attachment behavior. In addition, a minority of children who experienced severe early deprivation were classified as avoidant, secure, or dependent using conventional classification strategies, despite also exhibiting atypical patterns of attachment behaviors, and this was also more likely among children exposed to prolonged deprivation. The results raise both theoretical and methodological implications for attachment research on very deprived children.