Single-cell RNA sequencing identifies molecular targets associated with poor in vitro maturation performance of oocytes collected from ovarian stimulation.

STUDY QUESTION: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them? SUMMARY ANSWER: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes. WHAT IS KNOWN ALREADY: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM. STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-µM gallic acid on their maturation rate. MAIN RESULTS AND THE ROLE OF CHANCE: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/ß-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%. LARGE SCALE DATA: Raw data from this study can be accessed through GSE158539. LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.

Genome-wide association study in a Chinese population identifies a susceptibility locus for type 2 diabetes at 7q32 near PAX4.

AIMS/HYPOTHESIS: Most genetic variants identified for type 2 diabetes have been discovered in European populations. We performed genome-wide association studies (GWAS) in a Chinese population with the aim of identifying novel variants for type 2 diabetes in Asians. METHODS: We performed a meta-analysis of three GWAS comprising 684 patients with type 2 diabetes and 955 controls of Southern Han Chinese descent. We followed up the top signals in two independent Southern Han Chinese cohorts (totalling 10,383 cases and 6,974 controls), and performed in silico replication in multiple populations. RESULTS: We identified CDKN2A/B and four novel type 2 diabetes association signals with p < 1 × 10(-5) from the meta-analysis. Thirteen variants within these four loci were followed up in two independent Chinese cohorts, and rs10229583 at 7q32 was found to be associated with type 2 diabetes in a combined analysis of 11,067 cases and 7,929 controls (p meta = 2.6 × 10(-8); OR [95% CI] 1.18 [1.11, 1.25]). In silico replication revealed consistent associations across multiethnic groups, including five East Asian populations (p meta = 2.3 × 10(-10)) and a population of European descent (p = 8.6 × 10(-3)). The rs10229583 risk variant was associated with elevated fasting plasma glucose, impaired beta cell function in controls, and an earlier age at diagnosis for the cases. The novel variant lies within an islet-selective cluster of open regulatory elements. There was significant heterogeneity of effect between Han Chinese and individuals of European descent, Malaysians and Indians. CONCLUSIONS/INTERPRETATION: Our study identifies rs10229583 near PAX4 as a novel locus for type 2 diabetes in Chinese and other populations and provides new insights into the pathogenesis of type 2 diabetes.

The effect of telomere length, a marker of biological aging, on bone mineral density in elderly population.

UNLABELLED: Telomere length (TL), as a reflection of aging and inflammatory processes, may be associated with bone mineral density (BMD). This study examines the association between TL and BMD cross-sectionally and the rate of bone loss over a 4-year period in 1,867 Chinese elderly community living subjects. After adjusting for confounding factors, no association was observed with BMD or bone loss. The decline in BMD with aging is not reflected by corresponding changes in telomere length. INTRODUCTION: Bone mineral density (BMD) is influenced by the dynamics of aging, inflammatory, and bone remodeling processes. Telomere length (TL) is a reflection of the former two processes and may also be associated with bone loss. METHODS: Hip BMD was measured in 1,867 Chinese elderly community living subjects and the relationship between leukocyte TL measured using quantitative real-time polymerase chain reaction, and bone loss after 4 years was examined. RESULTS: Women had greater bone loss than men. In women, age of menopause, menarche, estrogen treatment/replacement therapy, and history of previous fracture were also among the significant covariates. However, in multivariate analyses, TL was not associated with BMD in either sex. CONCLUSIONS: TL was not associated with either baseline BMD or bone loss over 4 years and accounted for less than 1.6% of the baseline BMD.

Asthma and atopy are associated with chromosome 17q21 markers in Chinese children.

BACKGROUND: Single-nucleotide polymorphism (SNP)-based genome-wide association study revealed that markers on chromosome 17q21 were linked to childhood asthma but not atopy in Caucasians, with the strongest signal being detected for the SNP rs7216389 in the ORMDL3 gene. Such association was unknown in Chinese. This study delineated the allele and genotype frequencies of 10 SNPs at chromosome 17q21, and investigated the relationship between these SNPs and asthma and plasma IgE in southern Chinese children. METHODS: Asthmatic children and non-allergic controls were recruited from pediatric clinics. Their plasma total and aeroallergen-specific IgE concentrations were measured by immunoassay. Ten SNPs on 17q21 region were genotyped by multiplex SNaPshot, and their genotype associations with asthma traits analyzed using multivariate regression. RESULTS: 315 patients and 192 controls were enrolled. The allele frequency for C allele of rs7216389 varied significantly from 0.232 in our controls, 0.389 in Han Chinese to 0.536 in Caucasians. Asthma diagnosis was associated with rs11650680 and five other SNPs including rs7216389 (P = 0.019-0.034), whereas atopy was associated only with rs11650680 (P = 0.0004). Linear regression revealed the covariates for plasma total IgE to be significant for rs11650680 (P = 0.008-0.0002). Haplotypic associations were found with atopy and increased plasma total IgE, with the respective odds ratios and 95% confidence intervals for TTTCCGTT haplotype to be 0.21 and 0.09-0.52 (P = 0.0002) and 0.41 and 0.18-0.90 (P = 0.025). CONCLUSION: Childhood asthma and atopy are associated with chromosome 17q21 in Chinese, but such association may involve genes other than ORMDL3 in this region.

Polymorphisms in the C-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (SARS-CoV) infection.

BACKGROUND: Polymorphisms of CLEC4M have been associated with predisposition for infection by the severe acute respiratory syndrome coronavirus (SARS-CoV). DC-SIGNR, a C-type lectin encoded by CLEC4M, is a receptor for the virus. A variable number tandem repeat (VNTR) polymorphism in its neck region was recently associated with susceptibility to SARS infection. However, this association was controversial and was not supported by subsequent studies. Two explanations may account for this discrepancy: (1) there may be an unknown predisposition polymorphism located in the proximity which is linked to the VNTR; or (2) it was a spurious association due to unrecognised population structure in the VNTR. METHODS: We performed a comprehensively genetic association study on this C-type lectin gene cluster (FCER2, CLEC4G, CD209, and CLEC4M) at 19p13.3 by a tagging single nucleotide polymorphisms (SNPs) approach. RESULTS: 23 tagSNPs were genotyped in 181 SARS patients and 172 population controls. No significant association with disease predisposition was detected. Genetic variations in this cluster also did not predict disease prognosis. However, we detected a population stratification of the VNTR alleles in a sample of 1145 Han Chinese collected from different parts of China. CONCLUSION: The results indicated that the genetic predisposition allele was not found in this lectin gene cluster and population stratification might cause the previous positive association.

Telomeres and frailty.

Associations between telomere length and various chronic diseases associated with ageing have led to the suggestion that telomere length may be an ageing biomarker. At the clinical level, the suggestion of using measurements of frailty as a measure of biological ageing has also been suggested. This study examines the hypothesis that telomere shortening may form the biological basis for frailty, using data obtained from a health survey of 2000 men and women aged 65 years and over, living in the community, and followed up for 4 years to determine survival. Frailty was measured using the frailty index, a summation of deficits covering physical, psychological, and functional domains. Telomere length was measured in 976 men and 1030 women, using real-time quantitative polymerase chain reaction. Women were more frail than men but had longer telomere length. In men only, there was a negative association between telomere length and age and a positive association between frailty index and mortality after adjusting for age. There was no correlation between telomere length and frailty index in either sex. While telomere length may be a biomarker of cellular senescence, this relationship may not be extrapolated to the functional level represented by the frailty phenotype.

Study of gene-gene interactions for endophenotypic quantitative traits in Chinese asthmatic children.

BACKGROUND: Asthma is a complex disease resulting from interactions between multiple genes and environmental factors. Study of gene-gene interactions could provide insight into the pathophysiology of asthma. METHODS: We investigated the interactions among 18 single-nucleotide polymorphisms in eight candidate genes for plasma total immunoglobulin E (IgE) concentration and peripheral blood (PB) eosinophil count in 298 Chinese asthmatic children and 175 controls. Generalized multifactor dimensionality reduction and generalized linear model were used to analyze gene-gene interactions for the quantitative traits. RESULTS: A significant interaction was found between R130Q in IL13 and I50V in IL4RA for plasma total IgE concentration, with a cross-validation (CV) consistency of nine of 10 and a prediction error of 41.1% (P = 0.013). Plasma total IgE concentration was significantly higher in the high-risk than the low-risk groups (P < 0.0001). For PB eosinophil count, significant interaction was found between C-431T in TARC and RsaI_in2 in FCERIB, with a CV consistency of nine of 10 and a prediction error of 40.2% (P = 0.009). PB eosinophil count was significantly higher in the high-risk group than the low-risk groups (P < 0.0001). Generalized linear model also revealed significant gene-gene interaction for the above two endophenotypes with P = 0.013 for plasma total IgE concentration and P = 0.029 for PB eosinophil count respectively. CONCLUSIONS: Our data suggest significant interactions between IL13 and IL4RA for plasma total IgE concentration, and this is the first report to show significant interaction between TARC and FCERIB for PB eosinophil count in Chinese asthmatic children.

Narcolepsy in Southern Chinese patients: clinical characteristics, HLA typing and seasonality of birth.

OBJECTIVE: To report clinical characteristics, human leukocyte antigen (HLA) typing and seasonality of birth of a series of 54 Southern Chinese patients suffering from narcolepsy. METHODS: All subjects underwent detailed medical and psychiatric interviews and a standardised nocturnal polysomnogram followed by a daytime Multiple Sleep Latency Test. Each subject also completed a set of sleep questionnaires. HLA typing was performed in 91% of subjects. RESULTS: A total of 78% and 22% of patients were diagnosed with suffering from cataplectic and non-cataplectic narcolepsy, respectively. The majority (n = 47, 87%) of patients were referred to our sleep clinic for excessive daytime sleepiness (EDS). The cataplectic narcolepsy differed from non-cataplectic narcolepsy by having more rapid eye movement (REM)-related clinical symptoms (more sleep paralysis and sleep-related hallucination) and sleep disturbances (shorter REM latency), as well as tighter association with HLA DQB1*0602. A bi-modal peak pattern was observed at 11 and 39 years old. A similar bi-modal pattern also occurred for EDS and cataplexy. Excess winter births were observed for this series of patients. 81% of patients with cataplectic narcolepsy were DQB1*0602-positive. There were no differences between early- and late-onset cases in the association with positive DQB1*0602 (71.4% vs 60%). Narcolepsy had prominent pernicious effects on various social, academic, family and mental aspects in our patients. CONCLUSIONS: In our Southern Chinese narcolepsy series, bi-modal peak pattern of age of onset, excess winter birth and tight association of HLA DQB1*0602 with cataplectic narcolepsy were found.

Association study between adolescent idiopathic scoliosis and the DPP9 gene which is located in the candidate region identified by linkage analysis.

BACKGROUND: It has been recognised that genetic or hereditary factors may contribute to the aetiology of adolescent idiopathic scoliosis (AIS). Recently, two linkage analyses have identified 19p13.3 as the candidate region for AIS. The dipeptidyl peptidase 9 (DPP9) gene is located on chromosome 19p13.3. OBJECTIVE: To investigate whether DPP9 gene polymorphisms are associated with the occurrence or curve severity of AIS. METHODS: 571 girls with AIS and 236 normal controls were recruited. Using the Chinese data from the HapMap project, a set of tagging single-nucleotide polymorphisms (tagSNPs) were defined for the DPP9 gene. Five SNPs were genotyped by PCR restriction fragment length polymorphism. Statistical analysis of genotype frequencies between cases and controls was performed by the chi2 test. One-way analysis of variance was used to compare mean maximum Cobb angles with different genotypes in case-only analysis. RESULTS: Genotype frequencies were comparable between cases and controls for all five polymorphisms (p>0.05). The mean maximum Cobb angles of different genotypes were similar to each other for all five polymorphisms. CONCLUSIONS: The DPP9 gene is not associated with the occurrence or curve severity of AIS. It is neither a disease-predisposition nor a disease-modifying gene of AIS.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

RANTES G-401A polymorphism is associated with allergen sensitization and FEV1 in Chinese children.

G-401A polymorphism in RANTES promoter was associated with near-fatal asthma and atopic dermatitis in children. We studied whether gain-of-function mutations in RANTES gene were associated with asthma and atopy-related traits in Chinese children. Plasma total and aeroallergen-specific IgE concentrations were measured using micro-particle immunoassay and fluorescent enzyme immunoassay, respectively. Restriction fragment length polymorphism was used to genotype RANTES G-401A and C-28G. One hundred and twenty-nine asthmatic children and 66 controls were recruited. Their mean logarithmic plasma total IgE concentrations were 2.53 and 1.98, respectively (P<0.0001). RANTES G-401A was not associated with physician-diagnosed asthma (P = 0.408). However, RANTES G-401A allele was significantly associated with IgE sensitization to cat (odds ratio 2.35; 95% CI 1.15-4.77; P = 0.010). Those homozygous for -401A had higher plasma cat-specific IgE levels (P = 0.034). Subjects having -401A were also more likely to have mold-specific IgE (odds ratio 3.82; 95% CI 1.24-12.14; P = 0.007). On spirometry, those with -401A/ A had lower forced expiratory volume in 1-s (FEV1; P = 0.044). RANTES C-28G was not associated with any outcome in this study. In conclusion, the gain-of-function mutation at -401 of RANTES promoter is associated with sensitization to cat and mold allergens and FEV1 in Chinese children.

Distribution in allele frequencies of predisposition-to-atopy genotypes in Chinese children.

Asthma is one of the most common chronic diseases in childhood; it is caused by a complex interaction between genetic factors and exposure to environmental allergens and irritants. Previous studies using the candidate gene approach showed that asthma was linked to a number of susceptibility genetic loci in Caucasian subjects. There are, however, only a few studies on asthma predisposition genes in the Chinese population. We studied the distribution of allele frequencies of I50V for the interleukin-4 receptor, two polymorphisms in intron 2 and exon 7 for the high-affinity IgE receptor (Fc epsilon RI-beta), R16G and E27Q for the beta(2)-adrenoceptor,and R275Q (824G/A) for CC chemokine receptor 3 in Chinese children.Seventy-six patients, with a mean age of 10.6 years, and 70 age- and sex-matched controls, were studied. Significantly more subjects in the asthma group had specific IgE antibodies against environmental allergens (P < 0.0001; odds ratio, 9.82). Genotyping of the six genetic markers showed that none of the six polymorphisms was associated with asthma in this cohort. The allele frequencies of I50V, R16G, and E27Q in our population were similar to those published for Asian subjects but not Caucasians. The R275Q substitution was a rare finding in our study and in the published reports. Our results demonstrate ethnic differences in polymorphisms of atopy candidate genes. Additional studies involving larger samples are required to investigate the association between asthma or atopy and the genotypes studied to date in Chinese children.

Total daily energy expenditure in wasted chronic obstructive pulmonary disease patients.

OBJECTIVES: To investigate total daily energy expenditure in chronic obstructive pulmonary disease (COPD) patients during a rehabilitation programme. DESIGN: Observational study involving a case and a control group. SUBJECTS: Ten COPD patients (six with body mass index (BMI) <18.5 kg/m(2) and four with BMI >18.5 kg/m(2)) were evaluated for their energy expenditure profile. Four additional healthy age-matched volunteers were also included for methodology evaluation. INTERVENTIONS: Measurements of total daily energy expenditure (TEE), resting energy expenditure (REE) and diet-induced thermogenesis (DIT) and energy intake were undertaken by indirect calorimetry and bicarbonate-urea methods and dietary records. RESULTS: REE in COPD patients was not significantly different from that predicted by the Harris-Benedict equation. Before the exercise day the mean TEE was 1508 kcal/day and physical activity level (PAL as calculated by TEE/REE) was 1.52. On the exercise day the TEE increased to 1568 kcal/day and PAL was 1.60, but neither of these changes were significant. The energy cost of increased physical activity during rehabilitation exercise was estimated to be 191 kcal/day. No significant change was found in DIT between the two patient groups. However, overall energy balances were found to be negative (-363 kcal/day). CONCLUSION: The rehabilitation programme did not cause a significant energy demand in COPD patients. TEE in COPD patients was not greater than in free-living healthy subjects. Patients, who were underweight, did not have a higher TEE than patients with normal weight. This suggested that malnutrition in COPD patients was not due to an increased energy expenditure. On the other hand, a significant negative energy balance due to insufficient energy intake was found in seven out of 10 patients.

Unrelated umbilical cord blood transplantation in children: experience of the Hong Kong Red Cross Blood Transfusion Service.

OBJECTIVE: To review the outcome of unrelated umbilical cord blood transplantation in children using cord blood from the Hong Kong Red Cross Blood Transfusion Service. DESIGN: Retrospective study. PATIENTS: Records of eight patients who received unrelated umbilical cord blood transplants between 1999 and 2003 were reviewed. MAIN OUTCOME MEASURES: Engraftment of haematopoietic cells and graft-versus-host disease after transplantation. RESULTS: The median age of the patients was 4.9 years (range, 1.0-9.4 years). Five patients had acute leukaemia, one had non-Hodgkin's lymphoma, one had X-linked adrenoleukodystrophy, and one had mucolipidosis. The infused umbilical cord blood units contained a median of 6.7 x 10(7) /kg nucleated cells and 4.0 x 10(5) /kg CD34-positive cells. Neutrophil engraftment was achieved at a median of 13 days (range, 11-19 days) and, for seven patients, platelet engraftment was achieved at a median of 39 days (range, 24-98 days). Acute graft-versus-host disease occurred in all patients (grades I to III). One of the patients died because of encephalitis; of the other seven, five developed chronic graft-versus-host disease of the skin. At a median follow-up of 2 years, the four patients with leukaemia and the one with non-Hodgkin's lymphoma remained in continuous complete remission; the patient with adrenoleukodystrophy showed stabilisation of neurological condition. CONCLUSION: The Hong Kong Red Cross Blood Transfusion Service Cord Blood Bank stored cord blood units of good quality for transplantation, the outcome of which was comparable to that of bone marrow transplantation.

The Alu polymorphism of angiotensin I converting enzyme (ACE) and atherosclerosis, incident chronic diseases and mortality in an elderly Chinese population.

OBJECTIVE: We examined the contribution of ACE I/D polymorphism in a large Chinese population to four year change in ankle-brachial index (ABI), development of cardiovascular diseases and mortality in a prospective study adjusting for many confounding factors. METHOD: Data are drawn from a longitudinal study of 4000 community-living men and women aged 65 years and over, for which detailed information regarding lifestyle, chronic diseases, body mass index (BMI), ABI measurements and ACE polymorphisms were documented at baseline. During the fifth year of follow up, incident cardiovascular diseases, ABI, and mortality were documented, and related to ACE genotype adjusting for age, smoking, alcohol, dietary intake, physical activity, body mass index, and use of ACE inhibitors. RESULTS: Women with the D/D genotype had the greatest reduction in mean ABI after adjusting for confounding factors. D/D genotype was also more common among women who developed hypertension or myocardial infarction. However D/D genotype was associated with mortality only in men. CONCLUSION: In a Chinese elderly population, ACE polymorphism may be considered "deleterious" to longevity, the D/D genotype being associated with mortality, the atherosclerotic process, hypertension and myocardial infarction. There are gender differences in the relationship between D/D genotype and cardiovascular diseases and mortality may not be mediated by the atherosclerotic process alone.

A community-based study of insomnia in Hong Kong Chinese children: Prevalence, risk factors and familial aggregation.

OBJECTIVES: There has been limited data on familial aggregation of insomnia. We aimed to explore the prevalence, risk factors and familial aggregation of childhood insomnia with a large community-based sample. METHODS: A community-based epidemiologic study of sleep disorders was conducted among primary school children. Those children with at least one reported biological parent were recruited. A total of 5695 children (mean age 9.2; SD 1.8), 4939 of their reported biological mothers (mean age 38.9; SD 4.6) and 4289 of their reported biological fathers (mean age 43.3; SD 5.5) were studied. RESULTS: The rates of insomnia 3 times/week in the past 12 months were 4.0%, 12.8% and 9.7% for children, mothers and fathers, respectively. A robust familial aggregation of insomnia was found even after adjustment of the shared environmental and socio-demographic factors. There was a significant dose-response relationship among the children across their parental status from neither, fathers, mothers to both parents with insomnia [3.0%, 7.1%, 9.5% and 11.9%; with ORs (95% CIs)=2.48 (1.82-4.37) for fathers, 3.42 (2.55-4.59) for mothers and 4.42 (2.42-8.10) for both parents, respectively]. In addition, the frequency of insomniac symptoms of the parents also had a dose-response effect on the rate of insomnia of their children. CONCLUSIONS: Insomnia is a common problem in both children and their parents. A significant familial aggregation of childhood onset insomnia was seen in this study even after adjustment of the co-risk factors. There was a dose-response effect of parental insomnia on the rate of insomnia of their children with a slight predilection of maternal influences.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.