The developmental consequences of rare chromosome disorders : two case reports

Aim: As molecular and cytogenetic testing becomes increasingly sophisticated, more individuals are being diagnosed with rare chromosome disorders. Yet despite a burgeoning knowledge about biomedical aspects, little is known about implications for psychosocial development. The scant literature gives a general impression of deficits and adverse developmental outcomes. Method: Developmental data were obtained from two 16 year olds diagnosed with a rare chromosome disorder – a girl with 8p23.1 and a boy with 16q11.2q12.1. Measures of intellectual ability, academic achievement, and other aspects of functioning were administered at multiple time points from early childhood to adolescence. Results: Both adolescents experienced initial delays in motor and language development. Although the girl’s intelligence is assessed as being in the average range, she experiences difficulties with motor planning, spelling and writing. The boy has been diagnosed with a mild intellectual disability and demonstrates mild autistic features. Conclusions: The two case descriptions are in marked contrast to the published literature about these two chromosome anomalies. Both adolescents are developing much more positively than would be expected on the basis of the grim predictions of their paediatricians and the negative reports in the literature. It is concluded that, for most rare chromosome disorders, the range of possible developmental outcomes is currently unknown.

Supporting siblings of children with a rare chromosome disorder

Siblings play an important role in children’s learning and development. Interactions with brothers and sisters provide opportunities to learn about sharing and emotional reciprocity, to develop social skills, to express thoughts and feelings, and to practise resolving conflict. But for children whose brother or sister has a disability, such as a rare chromosome disorder, some of these sibling experiences may be different. Many parents worry about how their non-disabled child will be affected by the experience of living with a brother or sister with a disability, and a great deal of research has explored both the possible negative consequences and also the potential benefits for siblings. In this article, we summarise the research findings and provide suggestions for ways that parents can support the positive development and well-being of all their children.

Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 Pyrosequencing

Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10–5) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

Loss of breast epithelial marker hCLCA2 promotes epithelial to mesenchymal transition and indicates higher risk of metastasis

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.

Growth of confined cancer spheroids : a combined experimental marker, critical modelling approach

A critical step in the dissemination of ovarian cancer is the formation of multicellular spheroids from cells shed from the primary tumour. The objectives of this study were to apply bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer spheroids in vitro and simultaneously to build on a mathematical model describing the growth of multicellular spheroids in these biomimetic matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and grown for up to 4 weeks. Immunohistochemistry, imaging and growth analyses were used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel. The mathematical model was formulated as a free boundary problem in which each spheroid was treated as an incompressible porous medium. The functional forms used to describe the rates of cell proliferation and apoptosis were motivated by the experimental work and predictions of the mathematical model compared with the experimental output. This work aimed to establish whether it is possible to simulate solid tumour growth on the basis of data on spheroid size, cell proliferation and cell death within these spheroids. The mathematical model predictions were in agreement with the experimental data set and simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture duration and administration of a chemotherapeutic drug. Our computational model provides new perspectives on experimental results and has informed the design of new 3D studies of chemoresistance of multicellular cancer spheroids.

Resting metabolic rate is associated with hunger, self-determined meal size, and daily energy intake and may represent a marker for appetite

Background: There are strong logical reasons why energy expended in metabolism should influence the energy acquired in food-intake behavior. However, the relation has never been established, and it is not known why certain people experience hunger in the presence of large amounts of body energy. Objective: We investigated the effect of the resting metabolic rate (RMR) on objective measures of whole-day food intake and hunger. Design: We carried out a 12-wk intervention that involved 41 overweight and obese men and women [mean ± SD age: 43.1 ± 7.5 y; BMI (in kg/m2): 30.7 ± 3.9] who were tested under conditions of physical activity (sedentary or active) and dietary energy density (17 or 10 kJ/g). RMR, daily energy intake, meal size, and hunger were assessed within the same day and across each condition. Results: We obtained evidence that RMR is correlated with meal size and daily energy intake in overweight and obese individuals. Participants with high RMRs showed increased levels of hunger across the day (P < 0.0001) and greater food intake (P < 0.00001) than did individuals with lower RMRs. These effects were independent of sex and food energy density. The change in RMR was also related to energy intake (P < 0.0001). Conclusions: We propose that RMR (largely determined by fat-free mass) may be a marker of energy intake and could represent a physiologic signal for hunger. These results may have implications for additional research possibilities in appetite, energy homeostasis, and obesity. This trial was registered under international standard identification for controlled trials as ISRCTN47291569.

A strong quantitative trait locus for wing length on chromosome 2 in a wild population of great reed warblers

Wing length is a key character for essential behaviours related to bird flight such as migration and foraging. In the present study, we initiate the search for the genes underlying wing length in birds by studying a long-distance migrant, the great reed warbler (Acrocephalus arundinaceus). In this species wing length is an evolutionary interesting trait with pronounced latitudinal gradient and sex-specific selection regimes in local populations. We performed a quantitative trait locus (QTL) scan for wing length in great reed warblers using phenotypic, genotypic, pedigree and linkage map data from our long-term study population in Sweden. We applied the linkage analysis mapping method implemented in GRIDQTL (a new web-based software) and detected a genome-wide significant QTL for wing length on chromosome 2, to our knowledge, the first detected QTL in wild birds. The QTL extended over 25 cM and accounted for a substantial part (37%) of the phenotypic variance of the trait. A genome scan for tarsus length (a bodysize-related trait) did not show any signal, implying that the wing-length QTL on chromosome 2 was not associated with body size. Our results provide a first important step into understanding the genetic architecture of avian wing length, and give opportunities to study the evolutionary dynamics of wing length at the locus level. This journal is© 2010 The Royal Society.

Prediction of IBD based on population history for fine gene mapping

A novel multiple regression method (RM) is developed to predict identity-by-descent probabilities at a locus L (IBDL), among individuals without pedigree, given information on surrounding markers and population history. These IBDL probabilities are a function of the increase in linkage disequilibrium (LD) generated by drift in a homogeneous population over generations. Three parameters are sufficient to describe population history: effective population size (Ne), number of generations since foundation (T), and marker allele frequencies among founders (p). IBD L are used in a simulation study to map a quantitative trait locus (QTL) via variance component estimation. RM is compared to a coalescent method (CM) in terms of power and robustness of QTL detection. Differences between RM and CM are small but significant. For example, RM is more powerful than CM in dioecious populations, but not in monoecious populations. Moreover, RM is more robust than CM when marker phases are unknown or when there is complete LD among founders or Ne is wrong, and less robust when p is wrong. CM utilises all marker haplotype information, whereas RM utilises information contained in each individual marker and all possible marker pairs but not in higher order interactions. RM consists of a family of models encompassing four different population structures, and two ways of using marker information, which contrasts with the single model that must cater for all possible evolutionary scenarios in CM.

Mapping of multiple quantitative trait loci affecting bovine spongiform encephalopathy

A whole-genome scan was conducted to map quantitative trait loci (QTL) for BSE resistance or susceptibility. Cows from four half-sib families were included and 173 microsatellite markers were used to construct a 2835-cM (Kosambi) linkage map covering 29 autosomes and the pseudoautosomal region of the sex chromosome. Interval mapping by linear regression was applied and extended to a multiple-QTL analysis approach that used identified QTL on other chromosomes as cofactors to increase mapping power. In the multiple-QTL analysis, two genome-wide significant QTL (BTA17 and X/Y ps) and four genome-wide suggestive QTL (BTA1, 6, 13, and 19) were revealed. The QTL identified here using linkage analysis do not overlap with regions previously identified using TDT analysis. One factor that may explain the disparity between the results is that a more extensive data set was used in the present study. Furthermore, methodological differences between TDT and linkage analyses may affect the power of these approaches.

Genome-wide search for markers associated with bovine spongiform encephalopathy

A genome-wide search for markers associated with BSE incidence was performed by using Transmission-Disequilibrium Tests (TDTs). Significant segregation distortion, i.e., unequal transmission probabilities of alleles within a locus, was found for three marker loci on Chromosomes (Chrs) 5, 10, and 20. Although TDTs are robust to false associations owing to hidden population substructures, it cannot distinguish segregation distortion caused by a true association between a marker and bovine spongiform encephalopathy (BSE) from a population-wide distortion. An interaction test and a segregation distortion analysis in half-sib controls were used to disentangle these two alternative hypotheses. None of the markers showed any significant interaction between allele transmission rates and disease status, and only the marker on Chr 10 showed a significant segregation distortion in control individuals. Nevertheless, the control group may have been a mixture of resistant and susceptible but unchallenged individuals. When new genotypes were generated in the vicinity of these three markers, evidence for an association with BSE was confirmed for the locus on Chr 5.

Reduction in resting plasma granulysin as a marker of increased training load

Granulysin is a cytolytic granule protein released by natural killer cells and activated cytotoxic T lymphocytes. The influence of exercise training on circulating granulysin concentration is unknown, as is the relationship between granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. We examined changes in plasma granulysin concentration, natural killer cell number and cytotoxicity following acute exercise and different training loads. Fifteen highly trained male cyclists completed a baseline 40-km cycle time trial (TT401) followed by five weeks of normal training and a repeat time trial (TT402). The cyclists then completed four days of high intensity training followed by another time trial (TT403) on day five. Following one final week of normal training cyclists completed another time trial (TT404). Fasting venous blood was collected before and after each time trial to determine granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. Granulysin concentration increased significantly after each time trial (P<0.001). Pre-exercise granulysin concentration for TT403 was significantly lower than pre-exercise concentration for TT401 (-20.3 +/- 7.5%, P<0.026), TT402 (-16.7 +/- 4.3%, P<0.003) and 7T404 (-21 +/- 4.2%, P<0.001). Circulating natural killer cell numbers also increased significantly post-exercise for each time trial (P<0.001), however there was no significant difference across TT40 (P>0.05). Exercise did not significantly alter natural killer cell cytotoxicity on a per cell basis, and there were no significant differences between the four time trials. In conclusion, plasma granulysin concentration increases following moderate duration, strenuous exercise and is decreased in response to a short-term period of intensified training.

Stage-specific embryonic antigen-4 is not a marker for chondrogenic and osteogenic potential in cultured chondrocytes and mesenchymal progenitor cells

One important challenge for regenerative medicine is to produce a clinically relevant number of cells with consistent tissue-forming potential. Isolation and expansion of cells from skeletal tissues results in a heterogeneous population of cells with variable regenerative potential. A more consistent tissue formation could be achieved by identification and selection of potent progenitors based on cell surface molecules. In this study, we assessed the expression of stage-specific embryonic antigen-4 (SSEA-4), a classic marker of undifferentiated stem cells, and other surface markers in human articular chondrocytes (hACs), osteoblasts, and bone marrow-derived mesenchymal stromal cells (bmMSCs) and characterized their differentiation potential. Further, we sorted SSEA-4-expressing hACs and followed their potential to proliferate and to form cartilage in vitro. Cells isolated from cartilage and bone exhibited remarkably heterogeneous SSEA-4 expression profiles in expansion cultures. SSEA-4 expression levels increased up to approximately 5 population doublings, but decreased following further expansion and differentiation cultures; levels were not related to the proliferation state of the cells. Although SSEA-4-sorted chondrocytes showed a slightly better chondrogenic potential than their SSEA-4-negative counterparts, differences were insufficient to establish a link between SSEA-4 expression and chondrogenic potential. SSEA-4 levels in bmMSCs also did not correlate to the cells' chondrogenic and osteogenic potential in vitro. SSEA-4 is clearly expressed by subpopulations of proliferating somatic cells with a MSC-like phenotype. However, the predictive value of SSEA-4 as a specific marker of superior differentiation capacity in progenitor cell populations from adult human tissue and even its usefulness as a stem cell marker appears questionable.

Resequencing and fine-mapping of the chromosome 12q13-14 locus associated with multiple sclerosis refines the number of implicated genes

Multiple sclerosis (MS) is a common chronic inflammatory disease of the central nervous system. Susceptibility to the disease is affected by both environmental and genetic factors. Genetic factors include haplotypes in the histocompatibility complex (MHC) and over 50 non-MHC loci reported by genome-wide association studies. Amongst these, we previously reported polymorphisms in chromosome 12q13-14 with a protective effect in individuals of European descent. This locus spans 288 kb and contains 17 genes, including several candidate genes which have potentially significant pathogenic and therapeutic implications. In this study, we aimed to fine-map this locus. We have implemented a two-phase study: a variant discovery phase where we have used next-generation sequencing and two target-enrichment strategies [long-range polymerase chain reaction (PCR) and Nimblegen's solution phase hybridization capture] in pools of 25 samples; and a genotyping phase where we genotyped 712 variants in 3577 healthy controls and 3269 MS patients. This study confirmed the association (rs2069502, P = 9.9 × 10−11, OR = 0.787) and narrowed down the locus of association to an 86.5 kb region. Although the study was unable to pinpoint the key-associated variant, we have identified a 42 (genotyped and imputed) single-nucleotide polymorphism haplotype block likely to harbour the causal variant. No evidence of association at previously reported low-frequency variants in CYP27B1 was observed. As part of the study we compared variant discovery performance using two target-enrichment strategies. We concluded that our pools enriched with Nimblegen's solution phase hybridization capture had better sensitivity to detect true variants than the pools enriched with long-range PCR, whilst specificity was better in the long-range PCR-enriched pools compared with solution phase hybridization capture enriched pools; this result has important implications for the design of future fine-mapping studies.

An X chromosome association scan of the Norfolk Island genetic isolate provides evidence for a novel migraine susceptibility locus at Xq12

Migraine is a common and debilitating neurovascular disorder with a complex envirogenomic aetiology. Numerous studies have demonstrated a preponderance of women affected with migraine and previous pedigree linkage studies in our laboratory have identified susceptibility loci on chromosome Xq24-Xq28. In this study we have used the genetic isolate of Norfolk Island to further analyse the X chromosome for migraine susceptibility loci. An association approach was employed to analyse 14,124 SNPs spanning the entire X chromosome. Genotype data from 288 individuals comprising a large core-pedigree, of which 76 were affected with migraine, were analysed. Although no SNP reached chromosome-wide significance (empirical α = 1×10−5) ranking by P-value revealed two primary clusters of SNPs in the top 25. A 10 SNP cluster represents a novel migraine susceptibility locus at Xq12 whilst a 11 SNP cluster represents a previously identified migraine susceptibility locus at Xq27. The strongest association at Xq12 was seen for rs599958 (OR = 1.75, P = 8.92×10−4), whilst at Xq27 the strongest association was for rs6525667 (OR = 1.53, P = 1.65×10−4). Further analysis of SNPs at these loci was performed in 5,122 migraineurs from the Women’s Genome Health Study and provided additional evidence for association at the novel Xq12 locus (P<0.05). Overall, this study provides evidence for a novel migraine susceptibility locus on Xq12. The strongest effect SNP (rs102834, joint P = 1.63×10−5) is located within the 5′UTR of the HEPH gene, which is involved in iron homeostasis in the brain and may represent a novel pathway for involvement in migraine pathogenesis.