Available fragile site identification software packages (FSM and FSM3) assume that all chromosomal breaks occur independently. However, under a Mendelian model of inheritance, homozygosity at fragile loci implies pairwise correlation between homologous sites. We construct correlation models for chromosomal breakage data in situations where either partitioned break count totals (per-site single-break and doublebreak totals) are known or only overall break count totals are known. We derive a likelihood ratio test and Neyman’s C( α) test for correlation between homologs when partitioned break count totals are known and outline a likelihood ratio test for correlation using only break count totals. Our simulation studies indicate that the C( α) test using partitioned break count totals outperforms the other two tests for correlation in terms of both power and level. These studies further suggest that the power for detecting correlation is low when only break count totals are reported. Results of the C( α) test for correlation applied to chromosomal breakage data from 14 human subjects indicate that detection of correlation between homologous fragile sites is problematic due to sparseness of breakage data. Simulation studies of the FSM and FSM3 algorithms using parameter values typical for fragile site data demonstrate that neither algorithm is significantly affected by fragile site correlation. Comparison of simulated fragile site misclassification rates in the presence of zero-breakage data supports previous studies (Olmsted 1999) that suggested FSM has lower false-negative rates and FSM3 has lower false-positive rates.

DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-IIβ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer.

DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-II¿ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer. / European Union¿s 6th Framework project Newborns and genotoxic exposure risk (NewGeneris), British Council¿s United Kingdom Indian Education Research Initiative (UKIER)

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In an unselected series of mentally retarded children in the county of Västerbotten, Sweden, the annual incidence of children with severe mental retardation (SMR) (IQ &lt; 50) and alive at the age of one year decreased from 5.3 per 1,000 in 1959 - 1963 to 3.1 per 1,000 in 1967 -1970. This was mainly due to a decrease in the incidence of Down's syndrome. In parallel the proportion of mothers 35 years of age or more at the birth of the child decreased significantly. The prevalence of children with SMR in 1976 was 3.5 per 1,000. The main cause of the SMR was prenatal in 70 percent, perinatal in 8 percent and postnatal in 1 percent. The cause of the SMR was untraceable in 20 percent of the cases. Associated CNS-handicaps occurred in 52 percent of the cases. The annual incidence of mildly mentally retarded children (IQ 50 - 69) registered at the Bureau for Provision and Services for Mentally Retarded was 4.2 per 1,000 and the prevalence in 1979 was 3.8 per 1,000. The cause of the mild mental retardation (MMR) was untraceable in 43 percent. Prenatal causes were identified in as many as 43 percent. Perinatal causes were found in 7 percent and postnatal causes in 5 percent of the cases. Associated CNS-handicaps occurred in 30 percent of the cases.A syndrome of mental retardation with X-linked inheritance not recognized previously in Sweden was characterized clinically (mainly in boys, machro-orchidism, verbal disabilities) and cytogenetically (a fragile site on the X-chromosomes seen after cui turing in special folic acid deficient media) and found to have a prevalence of 6 percent in the population of severely mentally retarded boys. This makes this syndrome the next most common cause of SMR in boys after Down's syndrome. The chromosomal fragility was also identified in female carriers, which has implications for genetic counselling.Through identification of an untreated Phenylketonurie mother giving birth to five severely mentally retarded children, attention was focused on the risks for the fetus of the growing number of Phenylketonurie women identified neonatally and treated di etarily but untreated after the age of 10 - 15 years.Great improvement in intellectual and social ability was seen in a boy with phenylketonuria although the dietary treatment was not introduced until the age of eight years.Heavy irradiation of a fetus late in gestation caused mental retardation, microcephaly, stunted growth, and eye and teeth abnormalities, although such abnormalities are thought not to result from irradiation after 20 weeks of pregnancy. / <p>Endast s.1-71: sammanfattningen (kappan) i fulltexten.</p><p>Ej med i fulltexten s.72-145: 7 delar.</p> / digitalisering@umu

Made available in DSpace on 2015-03-26T13:42:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 852162 bytes, checksum: 18350a1fa68989163ff6494b7784f27a (MD5) Previous issue date: 2012-07-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fragile X syndrome is the most common inherited cause of intellectual disability with an estimated prevalence of 1 in 3600 males and 1 in 4000 to 6000 females, where it causes moderate to severe intellectual and social impairment together with syndromic features including large ears and head, long face and macroorchidism. It is most often is caused by expansion of a CGG trinucleotide repeat array in the 5' UT region of the FMR-1 gene at Xq27.3. This locus corresponds to a fragile site which can be observed in the metaphase chromosome following selective culture conditions. In this study we analyze two brothers with clinical indicative of fragile X syndrome. Peripheral blood lymphocytes were studied after 96 hrs culture periods at 37°C in RPMI 1640 (supplemented with 5% fetal serum bovine, 20 mM HEPES, 2mM glutamine and 1% M-PHA), LymphoGrow® and PB-MAX®. Two bottles for each medium was prepared for cultures with antagonists, and in each one 0,25 &#956;g/mL fluorodeoxyuridine (FUdR) or 8 &#956;g/mL methotrexate (Mtx) were added 72 hrs after the start of culture. Cultures were harvested after 95 hrs and 45 minutes using standard methods following exposure to 0,1 &#956;g/mL colcemid for 15 minutes. Slides were stained with Giemsa 5% for fra(X) screening and G banding was employed for confirmation of the Xq27.3 fragile site. Lymphocytes growing in RPMI 1640, LymphoGrow and PB-MAX responded equivalently to the addition of antagonists. According to the F test at the level of 5% probability, there were no significant differences between the average number of metaphases in the three mediums in combination with FUdR. Similarly, in the three mediums methotrexate treatment was very toxic and resulted in insufficient number of analyzable cells. No fra(X) were detected in the cultures with this treatment. A total of 208 metaphases for brother 1 and 205 for brother 2 were scanned of which 21% and 20%, respectively, were positive for fra(X). These results of fragile X expression in brothers are in agreement with the estimate expression in affected males ranging from 10-40% and corroborate those of other studies which reported that men of the same family tend to have similar frequencies of fra(X). The procedures for induction of fragile site and cytogenetic analysis showed to be adequate in order to identify the presence of FRAXA in the analyzed brothers with clinical suspicion of fragile X syndrome. / A Síndrome do X frágil é a principal causa de retardo mental hereditário com prevalência estimada de 1 em cada 3600 homens e 1 em cada 4000 a 6000 mulheres. Essa síndrome provoca comprometimento intelectual e social moderado a severo, associado com características como cabeça e orelhas grandes, rosto alongado e macroorquidismo. A causa mais frequente dessa desordem é a expansão do trinucleotídeo CGG localizado na região 5' UT do gene FMR-1, em Xq27.3. Esse loco corresponde a um sítio frágil que pode ser visualizado em cromossomos metafásicos sob condições seletivas de cultura. Neste estudo analisamos dois irmãos com indicativo clínico da Síndrome do X frágil. Linfócitos oriundos de sangue periférico foram avaliados após 96 h de cultura a 37°C em meio RPMI 1640 (suplementado com 5% de FBS, 20 mM de HEPES, 2 mM de glutamina e 1% M-PHA), LymphoGrow® e PB-MAX®. Para as culturas com antagonista, dois tubos de cada meio disponível foram preparados, e em cada um, 0,25 &#956;g/mL de fluorodeoxiuridina (FUdR) ou 8 &#956;g/mL de metotrexato (Mtx) foram adicionados 72 h após o início da cultura. As culturas foram colhidas após 95 h e 45 min utilizando-se métodos de rotina, após exposição a 0,1 &#956;g/mL de colcemide durante 15 min. As lâminas foram coradas com Giemsa 5% para rastreamento de cromossomos fra(X), e a técnica de bandeamento G foi empregada para a confirmação do sítio frágil na região Xq27.3. Os linfócitos cultivados nos meios RPMI 1640, LymphoGrow e PB-MAX responderam de forma equivalente à adição dos antagonistas. Não houve diferença significativa pelo teste F a 5% de probabilidade entre o número médio de metáfases observado nos três meios em combinação com FUdR. Do mesmo modo, nos três meios o tratamento com metotrexato foi muito tóxico e resultou em número insuficiente de células analisáveis. Nessas culturas com Mtx não foram detectados cromossomos fra(X). Um total de 208 metáfases para irmão 1 e 205 para o irmão 2 foram analisadas, das quais 21% e 20%, respectivamente, foram positivas para X frágil. Esses dados estão de acordo com a estimativa de expressão em homens afetados que varia de 10-40% e corroboram com os de outros estudos que reportaram que homens da mesma família tendem a apresentar frequências similares de fra(X). Os procedimentos para a indução de sítio frágil e análise citogenética mostraram-se adequados para identificação de FRAXA nos irmãos analisados com indicativo clínico da Síndrome do X frágil.