Copy number variation analysis detects novel candidate genes involved in follicular growth and oocyte maturation in a cohort of premature ovarian failure cases.

STUDY QUESTION: Can spontaneous premature ovarian failure (POF) patients derived from population-based biobanks reveal the association between copy number variations (CNVs) and POF? SUMMARY ANSWER: CNVs can hamper the functional capacity of ovaries by disrupting key genes and pathways essential for proper ovarian function. WHAT IS KNOWN ALREADY: POF is defined as the cessation of ovarian function before the age of 40 years. POF is a major reason for female infertility, although its cause remains largely unknown. STUDY DESIGN, SIZE, DURATION: The current retrospective CNV study included 301 spontaneous POF patients and 3188 control individuals registered between 2003 and 2014 at Estonian Genome Center at the University of Tartu (EGCUT) biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA samples from 301 spontaneous POF patients were genotyped by Illumina HumanCoreExome (258 samples) and HumanOmniExpress (43 samples) BeadChip arrays. Genotype and phenotype information was drawn from the EGCUT for the 3188 control population samples, previously genotyped with HumanCNV370 and HumanOmniExpress BeadChip arrays. All identified CNVs were subjected to functional enrichment studies for highlighting the POF pathogenesis. Real-time quantitative PCR was used to validate a subset of CNVs. Whole-exome sequencing was performed on six patients carrying hemizygous deletions that encompass genes essential for meiosis or folliculogenesis. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven novel microdeletions and microduplications that encompass genes relevant to POF were identified. For example, FMN2 (1q43) and SGOL2 (2q33.1) are essential for meiotic progression, while TBP (6q27), SCARB1 (12q24.31), BNC1 (15q25) and ARFGAP3 (22q13.2) are involved in follicular growth and oocyte maturation. The importance of recently discovered hemizygous microdeletions of meiotic genes SYCE1 (10q26.3) and CPEB1 (15q25.2) in POF patients was also corroborated. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. Anamnestic data obtained from population-based biobank lacked clinical, biological (hormone levels) or ultrasonographical data, and spontaneous POF was predicted retrospectively by excluding known extraovarian causes for premature menopause. WIDER IMPLICATIONS OF THE FINDINGS: The present study, with high number of spontaneous POF cases, provides novel data on associations between the genomic aberrations and premature menopause of ovarian cause and demonstrates that population-based biobanks are powerful source of biological samples and clinical data to reveal novel genetic lesions associated with human reproductive health and disease, including POF. STUDY FUNDING/COMPETING INTEREST: This study was supported by the Estonian Ministry of Education and Research (IUT20-43, IUT20-60, IUT34-16, SF0180027s10 and 9205), Enterprise Estonia (EU30020 and EU48695), Eureka's EUROSTARS programme (NOTED, EU41564), grants from European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, |EU324509) and Horizon 2020 innovation programme (WIDENLIFE, 692065), Academy of Finland and the Sigrid Juselius Foundation.

Clinical-scale in vitro expansion preserves biological characteristics of cardiac atrial appendage stem cells.

OBJECTIVES: Cardiac atrial appendage stem cells (CASCs) have recently emerged as an attractive candidate for cardiac regeneration after myocardial infarction. As with other cardiac stem cells, CASCs have to be expanded ex vivo to obtain clinically relevant cell numbers. However, foetal calf serum (FCS), which is routinely used for cell culturing, is unsuitable for clinical purposes, and influence of long-term in vitro culture on CASC behaviour is unknown. MATERIALS AND METHODS: We examined effects on CASC biology of prolonged expansion, and evaluated a culture protocol suitable for human use. RESULTS: In FCS-supplemented medium, CASCs could be kept in culture for 55.75 ± 3.63 days, before reaching senescence. Despite a small reduction in numbers of proliferating CASCs (1.37 ± 0.52% per passage) and signs of progressive telomere shortening (0.04 ± 0.02 kb per passage), their immunophenotype and myocardial differentiation potential remained unaffected during the entire culture period. The cells were successfully expanded in human platelet plasma supernatant, while maintaining their biological properties. CONCLUSIONS: We successfully developed a protocol for long-term culture, to obtain clinically relevant CASC numbers, while retaining their cardiogenic potential. These insights in CASC biology and optimization of a humanized platelet-based culture method are an important step towards clinical application of CASCs for cardiac regenerative medicine.

Suppressed increase in blood endothelial progenitor cell content as result of single exhaustive exercise bout in male revascularised coronary artery disease patients.

Endothelial progenitor cells (EPCs) significantly affect endothelial repair capacity and, hence, cardiovascular disease incidence. In healthy subjects, blood EPC content increases significantly as result of a single maximal exercise test, hereby stimulating endothelial repair capacity. It remains to be shown whether a single exercise positively affects blood EPCs in revascularised coronary artery disease (CAD) patients. From male revascularised CAD patients (n = 60) and healthy volunteers (n = 25) blood samples were collected before and immediately after a maximal cardiopulmonary exercise test. Blood samples were analyzed by optimised flow cytometry methodology for EPC content (CD34+, CD34+ CD133+, CD34+VEGFR2+, CD34+CD133+VEGFR2+, and CD34+CD133-VEGFR2+ cells) and compared between groups. CFU-Hill colonies were additionally assessed. As a result of a maximal exercise test, blood CD34+, CD34+VEGFR2+ (all EPCs), CD34+CD133+, and CD34+ CD133-VEGFR2+ (mature EPCs) cells increased significantly in CAD patients (p < 0.05), but less than in healthy subjects (p < 0.05, and p = 0.06 for CD34+VEGFR2+). CD34+CD133+VEGFR2+ cells (immature EPCs) did not change as result of exercise (p > 0.05). No changes in CFU-Hill colonies as result of exercise were observed. This study shows that blood mature EPCs (CD34+CD133-VEGFR2+) increase significantly as result of a single exercise bout in revascularised CAD patients, but with smaller magnitude compared to healthy subjects. Blood immature EPCs (CD34+CD133+VEGFR2+) did not change significantly as result of exercise.

Haplotype 4 of the multiple sclerosis-associated interleukin-7 receptor alpha gene influences the frequency of recent thymic emigrants.

The receptor for the homeostatic T cell cytokine interleukin-7 (IL-7Ralpha) has recently shown genetic association to multiple sclerosis (MS). To investigate the functional contribution of IL-7Ralpha polymorphisms to the pathogenesis of MS, we correlated the IL-7Ralpha haplotypes with different T cell parameters in a group of MS patients and healthy controls. We show that carriers of one of the four IL-7Ralpha haplotypes (Hap4) show a higher expression of IL-7Ralpha (CD127) on their CD4(+) T cells, compared with noncarriers (P=0.04). Moreover, Hap4 carriers possess higher frequencies of recent thymic emigrants (RTEs, CD31(+)) in both the regulatory T cell (Treg; P=0.007) and conventional T cell (Tconv) population (P=0.0001). This effect is most pronounced within the MS population (Treg, P=0.0077; Tconv, P=0.0007), whereas in healthy controls significance was only reached for Tconv (P=0.043; Treg, P=0.11). Because previous studies showed a decreased RTE-Treg frequency in MS patients compared to healthy subjects, we here conclude that this decrease is localized within the MS population of non-Hap4 carriers. In conclusion, our findings suggest that IL-7Ralpha polymorphisms can influence T cell development and homeostasis, and thereby contribute to the altered immune regulation associated with disease development in patients with MS.

Exploration of basal immune parameters in healthy infants receiving an infant milk formula supplemented with prebiotics.

This double-blind, randomized, placebo-controlled study, aimed to explore the effect of an infant milk formula (IMF) with 6 g/l short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS, ratio 9:1) on basal immune parameters in 215 healthy, term infants during the first 26 wk of life. After birth, the infants received breast milk or were randomized to receive an IMF with or without scGOS/lcFOS. Blood samples were collected at the age of 8 wk and 26 wk for the analysis of serum immunoglobulins, lymphocyte subpopulations, and cytokines. The scGOS/lcFOS group and the control group were compared in the statistical analysis. A breast fed group was included as a reference. In total, 187 Infants completed the study. No significant differences were observed between both formula groups in the different studied immune parameters at weeks 8 and 26. This explorative study indicates that supplementation of infant formula with a mixture of prebiotic oligosaccharides did not change the basal level of the measured parameters of the developing immune system in healthy infants with a balanced immune system during the first 6 months of life in comparison to feeding a standard infant formula and in comparison to exclusive breastfeeding.

Tetrabromo(2,6-dimethylpyridine-N)titanium(IV), a twinned crystal structure

The reaction of 2,6-dimethylpyridine with TiBr(4) affords the title compound, [TiBr(4)(C(7)H(9)N)], which is the first example of a neutral TiBr(4)L complex (L is a singly bonded ligand). The environment around the Ti atom can be described as a somewhat distorted trigonal bipyramid, with the nitrogen base occupying an equatorial position. The crystal was a non-merohedral twin.

The addition-reaction product of 1,1,1,4,4,4-hexachloro-1, 4-disilabutane with N-methylimidazole

The product of the addition reaction of 1,1,1,4,4,4-hexachloro-1, 4-disilabutane with N-methylimidazole is &mgr;-ethylene-C(1):C(2)-bis[dichlorotris(1-methylimidazole-N(3))sili con(IV)] dichloride, C(26)H(40)Cl(4)N(12)Si(2)(2+).2Cl(-). Two of the six Cl atoms are replaced by aromatic nitrogen bases and the coordination sphere of silicon is extended from four to six. The molecule is located on a crystallographic centre of inversion. The environment around the Si atom can be described as a slightly distorted octahedron with the Cl atoms occupying axial positions and the three N-methylimidazole ligands and the ethylene bridge in the equatorial plane.

Transcriptional activation capacity of the novel PLAG family of zinc finger proteins.

We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.