Retrospective cohort study and biobanking of patients treated for hemangioma in childhood – telomeres as biomarker of aging and radiation exposure

<p><b>Purpose:</b> Cohorts allowing joint epidemiological and biological analyses are essential for radiation risk assessment. The French Hemangioma Cohort (FHC), studied within the European project EpiRadBio, is one of the rare cohorts suitable for studying the effect of low dose radiation exposure (<100 mGy at organs), with a long-term follow-up. This highly homogeneous cohort consists of healthy individuals belonging to a normal population, except for the presence of skin hemangioma (age at exposure: between 6 months and 3 years of age). Published epidemiological studies have demonstrated that the risk of developing cancer is three times higher in the exposed individuals than in the general population. Here, we present the biobanking of samples (nucleated blood cells, cytogenetic slides of T and B lymphocytes) from the FHC and a primary feasibility study of biomarker analysis focusing on mean telomere length (MTL). Telomeres act as an internal clock, regulating the lifetime of the cell by their shortening during cell division. MTL is thus a biomarker of age. Many in vitro studies have linked MTL and radiosensitivity. The FHC will make it possible to discriminate between the effects of aging and radiation on this biomarker.</p> <p><b>Conclusion:</b> The establishment of a biobank of essentially healthy individuals (369 in total), exposed 40–70 years before, during their early childhood, is a logistical challenge. Even among those who previously participated to a self-questionnaire based study, the response rate was only 30%. The first biomarker to be studied was the MTL to discriminate age effects from those of radiation exposure. MTL showed significant variation within age groups (4–11 kb) in both the exposed and non-exposed groups. MTL within the limited age window (i.e. 40–73 year) examined, showed age-dependent changes of 46 bp/year, consistent with the age-dependent decline of 41 bp/year previously reported. We observed no significant changes in MTL according to the average active bone marrow dose. However, we were able to demonstrate that exposure to radiation causes the loss of cells with, on average, shorter telomeres, by applying a model in which both the heterogeneity of the individual dose received at the bone marrow and the heterogeneity of the intercellular distribution of MTL were taken into account.</p

DNA sequence of <i>att</i> and p<i>attP</i> sites.

<p>A) Wild type <i>attP</i> and <i>attB</i> sites. After recombination two hybrids sites are formed: <i>attL</i> and <i>attR</i>. B) Recombination between <i>attB</i> site and the human locus Xq22.1 This recombination generates a p<i>attR</i> which has been isolated by inverse PCR. Xq22.1 had been described previously as a human p<i>attP</i> by MP Calos et al., who isolated the same p<i>attR</i>.</p

Hypothetical model to explain the inversion of 4.8

<p>Step 1: Integration of a NILV mediated by phiC31-int into a p<i>attP</i> site. Step2: Recombination mediated by phiC31-int between the p<i>attL</i> generated during step 1 and another p<i>att</i> site located at 4 kb.</p

Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.

<p>Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.</p

Analysis strategies to detect the specific integrations mediated by phiC31-int.

<p>A) Illustration of the three mechanisms of the phiC31-int mediated integration of a NILV containing an <i>attB</i> sequence. According to the type of integration, the PCR results in three different profiles: - PCRs LTR+/<i>attB</i>− : integration type (1), specific integration. - PCRs LTR−/<i>attB</i>+: integration type (2), residual integration. - PCRs LTR+/<i>attB</i>+: integration type (3), illegitimate integration. P1/P1′ are the primers used for <i>attB</i> PCR and P2/P2′ are the primers used for LTR PCR. B) Schematic representations of the inverse PCR and the adapted inverse PCR strategies used to characterize phiC31-int integration sites.</p

Scheme of phiC31-int mediated recombination in bacterial host.

<p>PhiC31 integrase performs precise recombination between an <i>attB</i> site located in the <i>Streptomyces</i> genome and an <i>attP</i> site located on the phiC31 phage genome. The outcome is integration of the phage into the host genome.</p

Detection of recombination mediated by phiC31-int between an <i>attB</i> site contained into a NILV and a genomic <i>attP</i> site.

<p>A) Scheme of the DsRed2 PCR before and after the enzymatic restriction treatment. B) PCR DsRed2 results without restriction enzyme treatment. Lanes 1 to 3: cotransduction with CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lanes 4 to 6: cotransduction with <i>attB</i>-CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lane 7: <i>attB</i>-CMV-Neo. Lane 8: positive control generated by triple-transfection (CMV-phiC31-int, <i>attB</i>-CMV-Neo and CMV-<i>attP</i>-DsRed2). Lane 9: negative control without vector. Lane 10: negative control of PCR. C) PCR DsRed2 results after restriction enzyme treatment. Lanes are similar to figure B. D) Nested PCR from the product isolated from lane 6 to confirm the specificity of PCR DsRed2 amplification.</p

Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.

<p>Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.</p

Effect of NLS sequence on phiC31-int activity in NILV context.

<p>A) Cotransduction of NILVs CMV-PhiC31 and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). B) Cotransduction of NILVs CMV-PhiC31-NLS and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). No significant differences are observed between sample with or without a<i>ttB</i> sequence in the vector pTRIP-CMV-Neo. Satistics: two ways ANOVA with Bonferroni posttest (Prism 5).</p

Analysis of cell lines which constitutively expressed phiC31-int.

<p>A) PhiC31 RT-PCR on three different cell lines. HFi and Hi16 are derived from Hela cell line and TC1 from NIH-3T3 cell line. Control condition lane lacks RNA. B) PCR which detects LTR junctions or intact <i>attB</i> sites after transduction with a NILV <i>attB</i>-CMV-Neo.</p