A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Microarray analysis of primary epithelial and fibroblast cells in chronic rhinosinusitis without nasal polyps.

BACKGROUND: Chronic rhinosinusitis is an inflammatory condition with an as yet unknown pathophysiology. We aimed to detect clusters of differentially regulated genes in the epithelial and fibroblast cells of patients with Chronic Rhinosinusitis without nasal polyposis (CRSsNP) and healthy controls. METHODOLOGY: Carefully phenotyped CRSsNP and healthy control participants were recruited. Primary cultures of isolated epithelial and fibroblast cells were established. Whole transcriptome analysis of the cells was performed using microarrays and replicated with quantitative RT-PCR and immunohistochemistry. RESULTS: Fibroblast cells from CRSsNP patients showed a significant upregulation (more than 2x) of the transcription factor NFE2L3 when compared to healthy controls by microarray with multiple hypothesis testing correction, qRT-PCR and immunohistochemistry. CONCLUSIONS: Here we have utilized microarray analysis to search for differentially expressed genes in isolated patient derived epithelial and fibroblast cells. The transcription factor NFE2L3 has been shown to be upregulated in fibroblast cells consistent with increasing evidence that fibroblasts play a key role in tissue specific inflammation within the paranasal sinuses.

Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia.

High frequencies of blasts in primary acute lymphoblastic leukaemia (ALL) samples have the potential to induce leukaemia and to engraft mice. However, it is unclear how individual ALL cells each contribute to drive leukaemic development in a bulk transplant and the extent to which these blasts vary functionally. We used cellular barcoding as a fate mapping tool to track primograft ALL blasts in vivo. Our results show that high numbers of ALL founder cells contribute at similar frequencies to leukaemic propagation over serial transplants, without any clear evidence of clonal succession. These founder cells also exhibit equal capacity to home and engraft to different organs, although stochastic processes may alter the composition in restrictive niches. Our findings enhance the stochastic stem cell model of ALL by demonstrating equal functional abilities of singular ALL blasts and show that successful treatment strategies must eradicate the entire leukaemic cell population.

A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF-κB-dependent liver regeneration and cancer.

Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA.

Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc.

Only a small number of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater number of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.

The NF-κB subunit c-Rel regulates Bach2 tumour suppressor expression in B-cell lymphoma.

The REL gene, encoding the NF-κB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel-/- mice display significantly earlier lymphomagenesis in the c-Myc driven, Eµ-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Eµ-Myc lymphoma cells but, counterintuitively, c-rel-/- Eµ-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel-/- Eµ-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Eµ-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel-/- TCL1-Tg mice and RelA Thr505Ala mutant Eµ-Myc mice. Analysis of wild-type Eµ-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel-/- mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-κB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-κB/IκB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF-κB signalling in cancer.

Assessing readiness for change in the eating disorders: the psychometric properties of the readiness and motivation interview.

This study examined the psychometric properties of the Readiness and Motivation Interview (RMI), a symptom-specific measure of readiness and motivation for change in the eating disorders. For 4 symptom domains, the RMI assesses the extent to which individuals are in precontemplation, contemplation, and action/maintenance, and the extent to which change is made for internal versus external reasons. Ninety-nine individuals with eating disorders completed the RMI and measures to assess convergent, divergent, and criterion validity. RMI profiles revealed differences in readiness and motivation across symptom domains. The RMI demonstrated good reliability and construct validity, and RMI scores predicted anticipated difficulty of recovery activities, completion of recovery activities, decision to enroll in an intensive symptom-reduction program, and treatment dropout. The RMI may have important clinical applications by providing much-needed information on client readiness for action-oriented treatment.

Assessment of shape- and weight-based self-esteem in adolescents.

OBJECTIVES: The Adolescent version of the Shape and Weight-Based Self-Esteem (SAWBS-A) Inventory provides a contextual measure of the importance of shape and weight to overall feelings of self-worth. The psychometric properties of the adult SAWBS Inventory have been previously established. The present research examined the psychometric properties of this measure in adolescent females, and compared structural aspects of self-concept in symptomatic and nonsymptomatic individuals. METHOD: A school sample of 197 adolescents completed the SAWBS-A Inventory and measures to assess concurrent and discriminant validity. A subsample completed the SAWBS-A Inventory a second time, 1 week later. RESULTS: SAWBS-A scores were stable over 1 week and correlated with measures of eating disorder symptomatology. The scores also discriminated between adolescents reporting few or no disturbed eating symptoms and possible/probable cases. Comparison of self-esteem profiles in these two groups revealed that in addition to body, the groups differed in the extent to which facial appearance contributed to feelings of self-worth. CONCLUSIONS: The SAWBS-A Inventory may be useful in examining structural dimensions of self-esteem in adolescents.

Inhibited expression of negative emotions and interpersonal orientation in anorexia nervosa.

OBJECTIVE: This study examined inhibited expression of negative feelings and interpersonal orientation in women with anorexia nervosa. METHOD: Twenty-one women meeting DSM-IV criteria for anorexia nervosa were compared with 21 psychiatric and 21 normal control women matched on education. Two measures were used to assess inhibited expression of negative feelings and interpersonal orientation: the State-Trait Anger Expression Inventory assesses the suppression and expression of anger and the Silencing the Self Scale assesses four cognitive schemas involving the repression of needs and feelings to protect interpersonal relationships. RESULTS: Women with anorexia nervosa reported significantly higher scores on the four Silencing the Self schemas and on suppressed anger after controlling for age. These group differences were maintained for two of the cognitive schemas (Care and Silence) after controlling for depression, self-esteem, and global assessment of functioning. Inhibited expression of negative emotion and interpersonal orientation scores were also significantly related to cognitive and affective components of body image dissatisfaction and to trait and self-presentational dimensions of perfectionism. DISCUSSION: These findings are reviewed in the context of health psychology, as well as feminist and temperament theories. Implications for treatment are addressed.

Psychiatric comorbidity associated with eating disorder symptomatology among adolescents in the community.

OBJECTIVE: This study investigates psychiatric comorbidity associated with eating disorder symptomatology among adolescents in the community. METHOD: Four hundred three adolescents in the community were administered structured clinical interviews to assess mood, anxiety, eating, substance use, and personality disorders. RESULTS: Adolescents with dysthymia, panic and major depressive disorder were significantly more likely than those without these disorders to have an eating disorder. After controlling for the effects of other Axis I disorders and personality disorders, only dysthymia independently predicted the presence of an eating disorder. Several personality disorders were also associated with eating disorder symptoms. However, only obsessive-compulsive personality disorder predicted eating disorder symptoms after controlling for other personality disorders. CONCLUSION: Although previous research on adults has focused on the association between major depressive disorder and eating disorders, dysthymia may be more strongly associated with eating disorders among adolescents in the community. This association is not accounted for by psychiatric comorbidity.