Dehydroepiandrosterone at birth: Response to stress and relation to demographic, pregnancy and delivery factors.

Enhanced production of dehydroepiandrosterone (DHEA) by the foetal hypothalamic-pituitary-adrenal (HPA) axis enables maturational events critical for labour induction and neonatal adaptation. Despite knowledge of the interconnected nature of maternal and foetal physiology and dramatic changes in DHEA production after birth, few studies have examined DHEA levels in newborns and none have examined DHEA's response to acute stress. Understanding normative patterns of early DHEA activity is needed to accurately assess functioning of the biological stress system with relevance for health and development. The present study analysed DHEA concentrations and change after stress among 93 newborns and associations with pregnancy, delivery and demographic risk factors. Three saliva samples, collected prior to and following a blood draw stressor, were used to determine baseline and stress reactive DHEA levels. Mothers self-reported on health behaviours during pregnancy. Data on obstetric factors were obtained from medical records. DHEA levels declined from pre- to post-stressor assessments. Results also showed that post-stressor DHEA change was significantly associated with administration of medications used to treat pain and accelerate labour. However, there was no significant variation in DHEA pre-stress levels or change after stress as a function of time after birth. By capturing DHEA levels after birth, the present study provides a window into prenatal health of the HPA system. The study also advances knowledge of DHEA in newborns by providing data on reference levels and important covariates. This information on basic adrenal physiology provides a foundation that can be expanded on to enhance understanding of early hypothalamic-pituitary-adrenal axis activity.

Children's Relationship With Their Pet Dogs and OXTR Genotype Predict Child-Pet Interaction in an Experimental Setting.

Human-animal interaction (HAI) research has increasingly documented the important role of pet dogs in children's lives. The quality of interaction between children and their pet dogs, however, is likely influenced by individual differences among children as well as their perceived relationship with their pet dog. Ninety-seven children aged 7-12 years and their pet dogs participated in a laboratory protocol during which the child solicited interaction with their dog, from which time petting and gazing were recorded. Children reported on their perceived relationship with the pet dog via interview. Children provided saliva samples, from which a polymorphism in the oxytocin receptor, OXTR rs53576, which has long been implicated in social behavior, was genotyped. The results showed that OXTR genotype and children's perceived antagonism with the pet dog predicted the amount of petting, but not gazing, between children and their pet dogs. This research adds to the growing body of HAI research by documenting individual differences that may influence children's interactions with animals, which is key to research related to pet ownership and understanding factors that may impact therapeutic interventions involving HAI.

Putting a finger on the problem: Finger stick blood draw and immunization at the well-child exam elicit a cortisol response to stress among one-year-old children.

Research examining stress reactivity of the hypothalamic-pituitary-adrenocortical (HPA) axis in young children has historically been hampered by a lack of reliable methods to invoke a cortisol stress response. This report details an effective method of eliciting a cortisol rise in one-year-old children (N = 83) by modifying and combining two naturalistic stressors previously used with infants and children. Salivary cortisol levels were collected from children before and after a finger stick blood draw and immunizations performed during their one year well-child checkup at their pediatrician's office. Results indicated that the stressor was successful at eliciting a significant cortisol response. An extensive set of potential demographic and clinical confounds were also assessed in order to identify methodological considerations important in studies of infant cortisol. The stress paradigm presented here provides a promising alternative for studies of infant HPA activity to enable investigators to more effectively evaluate early functioning of the biological stress system during this developmentally important life stage.

BNDF methylation in mothers and newborns is associated with maternal exposure to war trauma.

BACKGROUND: The BDNF gene codes for brain-derived neurotrophic factor, a growth factor involved in neural development, cell differentiation, and synaptic plasticity. Present in both the brain and periphery, BDNF plays critical roles throughout the body and is essential for placental and fetal development. Rodent studies show that early life stress, including prenatal stress, broadly alters BDNF methylation, with presumed changes in gene expression. No studies have assessed prenatal exposure to maternal traumatic stress and BDNF methylation in humans. This study examined associations of prenatal exposure to maternal stress and BDNF methylation at CpG sites across the BDNF gene. RESULTS: Among 24 mothers and newborns in the eastern Democratic Republic of Congo, a region with extreme conflict and violence to women, maternal experiences of war trauma and chronic stress were associated with BDNF methylation in umbilical cord blood, placental tissue, and maternal venous blood. Associations of maternal stress and BDNF methylation showed high tissue specificity. The majority of significant associations were observed in putative transcription factor binding regions. CONCLUSIONS: This is the first study in humans to examine BDNF methylation in relation to prenatal exposure to maternal stress in three tissues simultaneously and the first in any mammalian species to report associations of prenatal stress and BDNF methylation in placental tissue. The findings add to the growing body of evidence highlighting the importance of considering epigenetic effects when examining the impacts of trauma and stress, not only for adults but also for offspring exposed via effects transmitted before birth.

Effect of Pet Dogs on Children's Perceived Stress and Cortisol Stress Response.

The present study tested whether pet dogs have stress-buffering effects for children during a validated laboratory-based protocol, the Trier Social Stress Test for Children (TSST-C). Participants were 101 children aged 7-12 years with their primary caregivers and pet dogs. Children were randomly assigned in the TSST-C to a pet present condition or one of two comparison conditions: parent present or no support figure present. Baseline, response, and recovery indices of perceived stress and cortisol levels were computed based on children's self-reported feelings of stress and salivary cortisol. Results indicated that in the alone (no social support) condition, children showed the expected rise for both perceived stress and cortisol response to stress. Pet dog presence significantly buffered the perceived stress response in comparison to children in the alone and parent present conditions. No main condition effect was observed for cortisol; however, for children experiencing the stressor with their pet present, lower cortisol response to stress was associated with more child-initiated petting and less dog proximity-seeking behavior. The results support the notion that pet dogs can provide socio-emotional benefits for children via stress buffering.

A high complex karyotype involving eleven chromosomes including three novel chromosomal aberrations and monoallelic loss of TP53 in case of follicular lymphoma transformed into B-cell lymphoblastic leukemia.

BACKGROUND: Follicular lymphoma (FL) is one of the most common B-cell non-Hodgkin's lymphoma (NHL). A subset of FL cases transform into more aggressive malignancies, most often to diffuse large B-cell lymphoma (DLBCL); in addition, lymphoblastic lymphoma and acute lymphoblastic leukemia (ALL) have also been rarely reported. The most common cytogenetic abnormalities associated with FL are translocation t(14;18)(q32;q21) with BCL2 rearrangements, present in 80-90% of all FL. However, that translocation alone is insufficient to cause FL and additional genomic events specifically leading to this kind of disease are still to be determined. The most frequently reported secondary changes are gains of chromosomes 7p or 7q, Xp, 12q and 18q, as well as losses on 6q and mutations within BCL2 and/or BCL6 genes. The presence of additional genomic aberrations, in particular 17p and 6q deletions is more frequent in grade 2 and 3 FL patients and correlated with shorter survival and a higher rate of transformation into DLBCL. CASE PRESENTATION: We describe here, an adult FL grade 2 patient that had transformed to B-ALL at diagnosis. Banding cytogenetics, refined by multi-color fluorescence in situ hybridization including array-proven multicolor banding revealed a unique complex karyotype involving eleven chromosomes, translocation t(X;20)(p21.3;q11.2), translocation t(3;20)(q26.2;q12), and a dicentric dic(17;18). Interestingly, the dicentric chromosome led to monosomy of the tumor suppressor gene TP53. The case had an immunophenotype consistent with follicular center cell lymphoma according to the World Health Organization (WHO) recommendations. CONCLUSIONS: To the best of our knowledge, a comparable adult FL grade 2 case that transformed to B-ALL associated with such a complex karyotype and loss of TP53 was not previously reported. Most of complex aberrations were found simultaneously in approximately 85% of studied malignant cells and the remained cells studied were non-clonal; mechanisms explaining this may be either multiple-step mechanisms or single step in sense of chromothripsis. TRIAL REGISTRATION: Identifying number: 3842. Registered 09 July 2012.

Complex translocation disrupting TCF4 and altering TCF4 isoform expression segregates as mild autosomal dominant intellectual disability.

BACKGROUND: Mutations of TCF4, which encodes a basic helix-loop-helix transcription factor, cause Pitt-Hopkins syndrome (PTHS) via multiple genetic mechanisms. TCF4 is a complex locus expressing multiple transcripts by alternative splicing and use of multiple promoters. To address the relationship between mutation of these transcripts and phenotype, we report a three-generation family segregating mild intellectual disability with a chromosomal translocation disrupting TCF4. RESULTS: Using whole genome sequencing, we detected a complex unbalanced karyotype disrupting TCF4 (46,XY,del(14)(q23.3q23.3)del(18)(q21.2q21.2)del(18)(q21.2q21.2)inv(18)(q21.2q21.2)t(14;18)(q23.3;q21.2)(14pter®14q23.3::18q21.2®18q21.2::18q21.1®18qter;18pter®18q21.2::14q23.3®14qter). Subsequent transcriptome sequencing, qRT-PCR and nCounter analyses revealed that cultured skin fibroblasts and peripheral blood had normal expression of genes along chromosomes 14 or 18 and no marked changes in expression of genes other than TCF4. Affected individuals had 12-33 fold higher mRNA levels of TCF4 than did unaffected controls or individuals with PTHS. Although the derivative chromosome generated a PLEKHG3-TCF4 fusion transcript, the increased levels of TCF4 mRNA arose from transcript variants originating distal to the translocation breakpoint, not from the fusion transcript. CONCLUSIONS: Although validation in additional patients is required, our findings suggest that the dysmorphic features and severe intellectual disability characteristic of PTHS are partially rescued by overexpression of those short TCF4 transcripts encoding a nuclear localization signal, a transcription activation domain, and the basic helix-loop-helix domain.

Chromosomes in a genome-wise order: evidence for metaphase architecture.

BACKGROUND: One fundamental finding of the last decade is that, besides the primary DNA sequence information there are several epigenetic "information-layers" like DNA-and histone modifications, chromatin packaging and, last but not least, the position of genes in the nucleus. RESULTS: We postulate that the functional genomic architecture is not restricted to the interphase of the cell cycle but can also be observed in the metaphase stage, when chromosomes are most condensed and microscopically visible. If so, it offers the unique opportunity to directly analyze the functional aspects of genomic architecture in different cells, species and diseases. Another aspect not directly accessible by molecular techniques is the genome merged from two different haploid parental genomes represented by the homologous chromosome sets. Our results show that there is not only a well-known and defined nuclear architecture in interphase but also in metaphase leading to a bilateral organization of the two haploid sets of chromosomes. Moreover, evidence is provided for the parental origin of the haploid grouping. CONCLUSIONS: From our findings we postulate an additional epigenetic information layer within the genome including the organization of homologous chromosomes and their parental origin which may now substantially change the landscape of genetics.

Prenatal Maternal Stress Predicts Methylation of Genes Regulating the Hypothalamic-Pituitary-Adrenocortical System in Mothers and Newborns in the Democratic Republic of Congo.

Exposure to stress early in life permanently shapes activity of the hypothalamic-pituitary-adrenocortical (HPA) axis and the brain. Prenatally, glucocorticoids pass through the placenta to the fetus with postnatal impacts on brain development, birth weight (BW), and HPA axis functioning. Little is known about the biological mechanisms by which prenatal stress affects postnatal functioning. This study addresses this gap by examining the effect of chronic stress and traumatic war-related stress on epigenetic changes in four key genes regulating the HPA axis in neonatal cord blood, placenta, and maternal blood: CRH, CRHBP, NR3C1, and FKBP5. Participants were 24 mother-newborn dyads in the conflict-ridden region of the eastern Democratic Republic of Congo. BW data were collected at delivery and maternal interviews were conducted to assess culturally relevant chronic and war-related stressors. Chronic stress and war trauma had widespread effects on HPA axis gene methylation, with significant effects observed at transcription factor binding (TFB) sites in all target genes tested. Some changes in methylation were unique to chronic or war stress, whereas others were observed across both stressor types. Moreover, stress exposures impacted maternal and fetal tissues differently, supporting theoretical models that stress impacts vary according to life phase. Methylation in several NR3C1 and CRH CpG sites, all located at TFB sites, was associated with BW. These findings suggest that prenatal stress exposure impacts development via epigenetic changes in HPA axis genes.

Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

Application of BAC-probes to visualize copy number variants (CNVs).

Copy number variations (CNVs) are structural variations of the human genome. These alterations result in variant copy numbers of certain stretches of DNA. In other words, some regions may be present in more or less copies than in a reference genome; however, these copy number changes do not have any impact on the phenotype. Also, CNVs may be extremely large and cytogenetically detectable or submicroscopic but still spanning several megabasepairs (Mb). In the recent years, array technology has identified especially the latter ones as so-called copy number variant (CNV) polymorphisms. These CNVs are detected in ~12 % of the human genome sequences and may comprise several hundred kilobasepairs. CNVs contribute significantly to the inter-individual differences in humans, and can range between 0.5 and 1.5 Mb amongst different genomes, well within the level of detection using cytogenetics techniques. Thus, they can be visualized by FISH using bacterial artificial chromosomes (BACs) as probes. Here we describe a method that enables discrimination of individual homologous chromosomes at the single cell level based on CNVs in the genome, called parental origin determination fluorescence in situ hybridization (POD-FISH). Possible fields of applications of this single cell-directed approach are in analyses of the parental origin of single chromosomes in inherited and acquired chromosomal aberrations.

A unique case of a discontinuous duplication 3q26.1-3q28 resulting from a segregation error of a maternal complex chromosomal rearrangement involving an insertion and an inversion.

Until now, few cases of partial trisomy of 3q due to segregation error of parental balanced translocation and segregation of a duplicated deficient product resulting from parental pericentric inversion have been reported so far. Only five cases of chromosomal insertion malsegregation involving 3q region are available yet, thus making it relatively rare. In this case report, we are presenting a unique case of discontinuous partial trisomy of 3q26.1-q28 region which resulted from a segregation error of two insertions involving 3q26.1 to 3q27.3 and 3q28 regions with ~21Mb and ~2Mb sizes, respectively. The maternally inherited insertion was cytogenetically characterized as der(8)(8pter→8p22::3q26→3q27.3::3q28→3q28::8p22→8qter) and the patient's major clinical features involved Dandy Walker malformation, sub-aortic ventricular septal defect, upslanting palpebral fissures, clinodactyly, hirsutism, and prominent forehead. Besides, a review of the literature involving cases with similar chromosomal imbalances and cases with "3q-duplication syndrome" is also provided.

Molecular cytogenetic evaluation of the efficacy of photodynamic therapy by indocyanine green in breast adenocarcinoma MCF-7 cells.

BACKGROUND: Photodynamic therapy (PDT) is used for the treatment of many types of predominantly epithelial cancers. Photosensitizer is taken up by fast growing tumor cells more actively than by other body cells and is activated by light, generating reactive oxygen species that cause cell death by necrosis or apoptosis. This study aimed to evaluate the efficacy of PDT with indocyanine green (ICG) through the investigation of TP53, HER-2 and TOP2A genes signals as breast cancer gene markers by interphase fluorescence in situ hybridization (nuc-FISH). METHODS: The photosynthetizer ICG (200 µM) was applied to breast cancer cell line MCF-7 cells (adenocarcinoma) in combination with laser irradiation (807 nm) exposure for 20 min and then incubated for 12, 24 and 48 h. Cell viability was evaluated using trypan blue. The signals for nuc-FISH was investigated and counted for probes specific for the genes TP53 (17p13), HER-2 (17q11.2-q12), and TOP2A (17q21-q22), and BAC-probes RP11-746M1 in 17p11.2 and RP11-403E9 in 17q11.2. RESULTS: The cell viability of MCF-7 did not reduced significantly when the cells were treated with ICG (200 µM) or exposed to laser irradiation for 20 min followed by incubation for 24 h. ICG/PDT treatment with laser irradiation exposure for 20 min reduced the cell viability after incubating cells for 12, 24 and 48 h highly significantly in a time dependent manner. For nuc-FISH analysis, TP53, HER-2, TOP2A, RP11-746M1 and RP11-403E9 signals did not reduce or increase in a significant manner when the cells were treated with ICG or exposed to laser irradiation for 20 min then incubated for 24h. PDT enhanced amplification of TP53 signals from nuc ish 17p13(TP53×2) to nuc ish 17p13(TP53×3) or nuc ish 17p13(TP53×4). However, the signals of HER-2 gene, TOP2A gene and BAC probes were reduced highly significantly when MCF-7 cells were treated with PDT with all time intervals. CONCLUSION: ICG/PDT and laser induced cytotoxic effect in MCF-7 cells. Also, PDT enhanced TP53 gene amplification, and reduced HER-2, TOP2A, and BAC probes RP11-746M1 and RP11-403E9 signals. Therefore ICG/PDT can be used for breast cancer treatment. It has the potential to induce apoptotic effect and reduce HER-2 and TOP2A genes propagation. Further in vivo studies are needed to evaluate ICG/PDT as a promising therapeutic approach for breast cancer.

Heteromorphic variants of chromosome 9.

BACKGROUND: Heterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers. RESULTS: In this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes. CONCLUSIONS: Based on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.

Human ring chromosomes and small supernumerary marker chromosomes-do they have telomeres?

Ring chromosomes and small supernumerary marker chromosomes (sSMC) are enigmatic types of derivative chromosomes, in which the telomeres are thought to play a crucial role in their formation and stabilization. Considering that there are only a few studies that evaluate the presence of telomeric sequences in ring chromosomes and on sSMC, here, we analyzed 14 ring chromosomes and 29 sSMC for the presence of telomeric sequences through fluorescence in situ hybridization (FISH). The results showed that ring chromosomes can actually fall into two groups: the ones with or without telomeres. Additionally, telomeric signals were detectable at both ends of centric and neocentric sSMC with inverted duplication shape, as well as in complex sSMC. Apart from that, generally both ring- and centric minute-shaped sSMC did not present telomeric sequences neither detectable by FISH nor by a second protein-directed immunohistochemical approach. However, the fact that telomeres are absent does not automatically mean that the sSMC has a ring shape, as often deduced in the previous literature. Overall, the results obtained by FISH studies directed against telomeres need to be checked carefully by other approaches.

Microdeletion and microduplication syndromes.

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted "genomic disorders" or "contiguous gene syndromes" is given.

Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats.

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.