Semi-quantitative Multiplex PCR Research Articles

Copy-number analysis of Y-linked loci in young men with non-obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y.

PurposeMosaic loss of chromosome Y (mLOY) is a common feature in elderly men. If mLOY can also occur in young men, it may lead to spermatogenic failure due to loss of spermatogenic genes. Indeed, previous studies detected the 45,X/46,XY karyotype in a few young men with spermatogenic failure. The present study aimed to clarify the frequency of cryptic mLOY in reproductive‐aged men with spermatogenic failure.MethodsWe studied 198 men at ages 24‐55 years who presented with etiology‐unknown non‐obstructive azoospermia. Prior this study, these patients underwent G‐banding analysis for 20 leukocytes and were found to have 46,XY karyotype. We analyzed copy numbers of chromosome Y in blood cells by using semi‐quantitative multiplex PCR for AMELY/AMELX, array‐based comparative genomic hybridization (CGH) for the AMELY locus, and droplet digital PCR for SRY, USP9Y, and UTY.ResultsMultiplex PCR showed borderline low AMELY/AMELX ratios in three patients. However, for the three patients, CGH excluded deletion of the AMELY locus, and droplet digital PCR suggested preserved copy numbers of all tested loci.ConclusionThis study highlights the rarity of leukocyte mLOY in reproductive‐aged men with spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.

Human Adenovirus (HAdV) Viremia in Immunocompetent Children with HAdV Infection in Respiratory Specimens: Does Viremia Predict Severity of Illness?

BackgroundThe interpretation of HAdV PCR from upper respiratory tract (RT) specimens can be challenging due to prolonged low grade viral shedding. We hypothesized that HAdV detection in the blood (viremia) is more common in acute HAdV infection with high respiratory viral burden (VB) compared with those with low VB in the RT. We sought to determine the frequency of HAdV viremia in immunocompetent children who have detectable HAdV in the RT.MethodsWe prospectively identified + HAdV in RT specimens from emergency department or inpatients using semi-quantitative real-time PCR (Ct <40) or multiplex respiratory viral PCR (FILMARRAY RESPIRATORY PANEL v1.7) and prospectively collected available whole blood from 8/2013 to 2/2015. Blood was considered positive for HAdV if Ct was <40 in whole blood. We compared virologic, including HAdV type from the RT and blood, and clinical characteristics between viremic and non-viremic groups using Mann–Whitney or chi-square as appropriate.ResultsThere were 196 unique patients with + HAdV in RT specimens as well as available blood for PCR (median age=1.3 years old). Blood and RT samples were obtained on the same calendar day in 78% of patients. Among these 196 patients, 163 (83%) were hospitalized and 58 (36%) were admitted to PICU. HAdV was detected in the blood in 33% of patients. Upper respiratory tract infections were more common (P = 0.026) and the duration of fever at the time of enrollment was longer in the viremia group (3 vs. 2 days, P = 0.043). There was no difference in ICU admission between two groups. Coinfections with bacterial pathogens from sterile sites were only found in the non-viremic group (4%); these included S. aureus or pneumococcal bacteremia, E. coli urinary tract infections, or pneumococcal pneumonia. HAdV VB in RT were significantly higher in the viremia group (Ct median 25.01 vs.. 36.38, P < 0.0001). Species C was more predominant in the viremia group compared with non C (A, B/E, D, F) (P = 0.018). RT type was 100% concordant with blood type.ConclusionHAdV viremia is relatively common in immunocompetent children with HAdV infection in the RT. Subjects with viremia had significantly higher VB in the RT, but this didn’t seem to be correlated with disease severity. HAdV viremia may be useful tool to add further evidence of acute HAdV infection.Disclosures A. Leber, BioFIre Diagnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

AB096. Molecular diagnosis of aniridia or WAGR syndrome using a simple DHPLC-based semi-quantitative multiplex PCR for detection of PAX6 large deletion

AB096. Molecular diagnosis of aniridia or WAGR syndrome using a simple DHPLC-based semi-quantitative multiplex PCR for detection of PAX6 large deletion

Large Deletions of TSPAN12 Cause Familial Exudative Vitreoretinopathy (FEVR).

Familial exudative vitreoretinopathy (FEVR) is a rare, hereditary visual disorder. The gene TSPAN12 is associated with autosomal dominant inheritance of FEVR. The prevalence and impact of large deletions/duplications of TSPAN12 on FEVR patients is unknown. To glean better insight of TSPAN12 on FEVR pathology, herein, we describe three FEVR patients with TSPAN12 deletions. Thirty-three Korean FEVR patients, who previously screened negative for TSPAN12 mutations, mutations in other FEVR-associated genes such as NDP, FZD4, LRP5, and large deletions and duplications of NDP, FZD4, and LRP5, were selected for TSPAN12 large deletion and duplication analyses. Semiquantitative multiplex PCR for TSPAN12 gene dosage analyses were performed, followed by droplet digital PCR (ddPCR) for validation. Among the 33 patients, three patients were confirmed to carry large TSPAN12 deletions. Two of them had whole-gene deletions of TSPAN12, and the other patient possessed a deletion of TSPAN12 in exon 4. FEVR severity detected in these patients was not more severe than in a patient with TSPAN12 point mutation. Regarding previously reported proportions of FEVR-associated genes contributing to the disorder's autosomal dominant inheritance pattern in Korea, we determined that patients with TSPAN12 large deletions were more common than patients with single nucleotide variants in TSPAN12. Evaluating TSPAN12 large deletions and duplications should be considered in FEVR screening and diagnosis as well as in routine genetic workups for FEVR patients.

A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis.

A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus crispatus and Lactobacillus iners. Together with a short questionnaire, a vaginal swab for PCR and vaginal smear for microscopy were taken by their general practitioner or gynaecologist. Data from 151 women (median age 32) were available. Nugent Score (NS) was used to classify the samples and 83 samples were classified as normal (NS 0–3), 13 as intermediate (NS 4–6), and 55 as bacterial vaginosis (NS 7–10). In women with a NS of 7–10, PCR detected Gardnerella vaginalis, Atopobium vaginae and Megasphaera phylotype 1 in respectively, 96 %, 87 % and 60 %, whereas in women with a NS of 1–3 these species were detected in 27 %, 6 % and 2 % (P <0.001). A ratio of Lactobacillus crispatus over Lactobacillus iners of <1 (as calculated from the quantification cycle value (Cq)) was present in women with a NS of 7–10 in 66 % versus 33 % in women with a NS of 1–3 (P <0.001). The BV-PCR displayed a sensitivity of 92 % and specificity of 96 % with a positive predictive value of 94 % and a negative predictive value of 95 %. The Lactobacillus-index improved the correct classification of samples where only one of the other bacterial species was detected. Compared to the Nugent Score this multiplex qPCR offers a convenient tool for performing observer independent diagnosis of BV.

Copy number variants in Italian Large White pigs detected using high-density single nucleotide polymorphisms and their association with back fat thickness.

The aim of this study was to identify copy number variants (CNVs) in Italian Large White pigs and test them for association with back fat thickness (BFT). Within a population of 12 000 performance-tested pigs, two groups of animals with extreme and divergent BFT estimated breeding values (EBVs; 147 with negative and 150 with positive EBVs) were genotyped with the Illumina Porcine SNP60 BeadChip. CNVs were detected with PENNCNV software. We identified a total of 4146 CNV events in 170 copy number variation regions (CNVRs) located on 15 porcine autosomes. Validation of detected CNVRs was carried out (i) by comparing CNVRs already detected by other studies and (ii) by semiquantitative fluorescent multiplex (SQFM) PCR of a few CNVRs. Most of CNVRs detected in Italian Large White pigs (71.2%) were already reported in other pig breeds/populations, and 82.1% of the CNV events detected by PENNCNV were confirmed by SQFM PCR. For each CNVR, we compared the occurrence of CNV events between the pigs of the high and low BFT EBV tails. Sixteen regions showed significance at P < 0.10, and seven were significant at P < 0.05 but were not significant after Bonferroni correction (Fisher's exact test). These results indicated that CNVs could explain a limited fraction of the genetic variability of fat deposition in Italian Large White pigs. However, it was interesting to note that one of these CNVRs encompassed the ZPLD1 gene. In humans, a rare CNV event including this gene is associated with obesity. Studies identifying CNVs in pigs could assist in elucidating the genetic mechanisms underlying human obesity.

Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV Assay for Human Papillomavirus Genotyping

The Anyplex II HPV28 (H28; Seegene) is a new semiquantitative real-time multiplex PCR assay for screening and genotyping 28 human papillomaviruses (HPV) in only 2 reaction wells. H28 was compared to the PGMY-CHUV assay (PG) with 309 archival DNA samples from cervical smears collected over 8 years in our laboratory. H28 and PG were fully concordant at the genotypic level on 228 (73.8%) out of 309 samples: 27 HPV negative and 201 HPV positive. The 201 fully concordant positive samples corresponded to single infections (n = 145) and to multiple infections (2 genotypes, n = 38; 3 to 5 genotypes, n = 18). The remaining 81 samples (26.2%) were either partially concordant (n = 64, 20.7%) or fully discordant (n = 17, 5.5%). While genotype-specific agreement was nearly perfect (κ = 0.877), HPV51 was significantly less well detected by H28 and the converse was observed for HPV40, -42, -54, and -68. Sequencing of PG amplicons confirmed HPV51 discordants and suggested the involvement of a possibly local HPV51 subtype. Mismatches in the PGMY09 primers to HPV68a explained most of the HPV68 discordants, confirming the specificity of H28 toward HPV68. With PG as a reference, the sensitivity and specificity of H28 were 93.4% and 99.0%, respectively. Considering H28 as a reference, the sensitivity and specificity of PG were 83.8% and 99.6%, respectively. H28 is a very sensitive and specific HPV genotyping assay suitable for research and clinical use as an adjunct to a clinically validated test. H28 semiquantitative readout ought to be evaluated for primary cervical cancer screening.

The Immunologic Properties of Undifferentiated Stem Cells from Human Exfoliated Deciduous Teeth (SHED) and Its Potential Application in Bone Regeneration

Introduction: Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic cells capable of differentiating into osteoblasts and inducing bone formation. It is a potential alternative for stem cell bone regeneration therapy. However, stem cell therapy carries the risk of immune rejection mediated by inflammatory cytokines of the human defense system. Objective: This preliminary research studies the interaction between SHED and the immune system by determining the inflammatory cytokines profile and osteogenic potential of SHED. Methods: Human fetal osteoblasts (hFOb) cell line and isolated SHED were cultured and total RNA was extracted, followed by reverse transcription cDNA synthesis. Semi-quantitative reverse transcription PCR and Multiplex PCR were performed to detect the expression levels of OPG/RANKL and TNF-α, IL-1β, IL-6, IL-8 and TGF-β in both cell types. Results: Analysis showed that SHED expressed significantly lower amounts of IL-1β, IL-6, and IL-8 compared to hFOB. IL-1β is a potent bone-resorbing factor, while IL-6 and IL-8 induce osteoclastogenesis and osteolysis respectively. SHED did not express TNF-α which stimulates osteoclastic activity. SHED demonstrated high OPG/RANKL ratio, in contrast with that of marrow stem cells described in previous studies. Our findings suggest that SHED may have improved immunomodulatory profile in terms of promoting relatively lower inflammatory reaction during transplant and enhancing bone regeneration. Conclusion: SHED has a potential to be a good source of osteoblasts for bone regeneration therapy. Further studies on the immunomodulatory properties of SHED-derived osteoblasts are necessary to enable stem cell therapy in immunocompetent hosts

Comparative microscopic and molecular analysis of Thatcher near‐isogenic lines with wheat leaf rust resistance genes Lr2a, Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races

Thatcher near‐isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticina‐ and wheat‐specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P. triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45·5–51·5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.

P3.13 Immune Status Stratification of Metastatic Breast Cancer Patients: Lympho-Divpenia Predicts Overall Survival

ABSTRACT Background Lymphopenia ( 1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor -beta chain (TCR-β), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n = 66) and in a validation series (cohort B, n = 67). A prognostic score, defined NDL (Number & Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results Lymphopenia ( 1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity >33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A + B) (p = 0.005) along with triple negative tumors (p = 0.011) and hemoglobin level (11.5 g/dL) (p = 0.009). Conclusion NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

Lymphopenia combined with low TCR diversity to predict overall survival in metastatic breast cancer patients.

10562 Background: Lymphopenia (&lt;1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor beta chain (TCR-ß), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods: Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n=66) and in a validation series (cohort B, n=67). A prognostic score, defined NDL (Number &amp; Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results: Lymphopenia (&lt;1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity &gt;33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A+B) (p=0.005) along with triple negative tumors (p=0.011) and hemoglobin level (11.5 g/dL) (p=0.009). Conclusions: NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

Evaluation of three next generation sequencing platforms for immune repertoire sequencing (58.10)

Abstract The advent of next generation sequencing (NGS) techniques, combined with a semi-quantitative multiplex PCR method, enables us to comprehensively profile the lymphocyte receptor diversity (immune repertoire) of the entire collection of B or T cells from a particular sample. Different NGS platforms vary in read length, throughput, sequencing error rate and error pattern, hands-on time, turnaround time and price. Three popular NGS techniques (Illumina HiSeq, 454 FLX and Ion Torrent) were employed to sequence amplicons from T cell receptor clones generated with arm-PCR in order to evaluate the usage of those techniques on immune repertoire sequencing. The error pattern and rate of the three techniques in the context of T cell receptor sequences were profiled. Based on the differences of the three techniques, we discuss the usage of each NGS platform in the context of immune repertoire sequencing.

Beta-Globin Cluster Cis-ACTING Sequence and BCL11A Variants in the Control of Hemoglobin F Expression

Abstract 1091Fetal hemoglobin (Hb F: α2γ2.) is the most important modulating factor of sickle cell disease(SCD) and beta-major thalassemias (β°thal). Due to the expected benefit from an active control of gamma gene expression, studies of the regulation of Hb F expression are very intense with the recent focus on BCl11 A, a transacting factors strongly involved in the control of its expression. Studies have subsequently established that Bcl 11A is a repressor of HbF expression through different mechanisms including a direct action “in trans” on the β-globin locus. Our work aims to look for Bcl 11A coding sequence’s variations in a cohort of homozygous β°thal patients whose clinical status is less severe than that expected from their globin genotype because of high Hb F level (12 patients). Interest has also been given to a cohort of patients (12 patients) displaying a small deletions of the Ψβ-δ intergenic region for which it has been noted significant Hb F rate differences in possible correlation with a Bcl 11A binding site located1.5 Kb 5 ’to the δ gene previously described (Sankaran, 2008). Methods:Sequencing of Bcl 11A (promoter, XL and L isoforms exons and flanking regions) was performed and the determination of the four main SNPs cited in the literature as predictors of the rate of HbF: rs4671393 and rs11886868 (Bcl 11A), rs9399137 (HSB1L-MYB), and rs7482144-XmnI (β-globin cluster), was carried out. The precise study of deletions was performed using a custom CGH array chip and the subsequent determination of breakpoints. In both patients’ cohorts, the absence of the deletion of one BCL11A allele was checked by a tailored semi-quantitative Multiplex PCR, and the hypothesis of a sequence variation inside the “Sankaran region” was screened using a High Resolution Melting procedure (HRM). Results and discussion:Analysis of BCL11A: We have not found mutation inside the coding sequence. However, A “CCG” triplet insertion within a CCG-rich region located in the 5'UTR, immediately 5' to the ATG codon was observed in three patients. That triplet insertion was not found in the other patients of our cohort of 12 patients with high expression of Hb F, and neither in another cohort of high F patients (23 patients). It was also absent in low Hb F patient ( 15) or in controls (50 patients not known to have a red cell disease), highlighting the rarity of this insertion. The effects on transcription or RNA translation could not be predicted by “in silico” analysis.The deletion analysis: We found in this cohort patients two kind of deletions: a first group where the 5’ breakpoint of the deletion is located close to the δ-globin gene and for which the presumed BCL11A binding domain was not removed and a second group for which the 5’ deletion breakpoint extends 5’ and removes that region. Interestingly the second group has a higher Hb F expression calculated as the absolute level of Hb f (Hb value * Hb F %) than the first group with a ratio of 2.54. That result highlight the role of this domain for Hb F control. We, also, cannot found sequence variation in that domain using HRM analysis. In conclusion:The presence of BCL 11A binding sites immediately 5’ of d gene appears to be necessary for Hb F control. However, investigating the role of Bcl 11A in Hb F dysregulation (ie: the absence of down regulation) either by looking at protein variants or at variations in the supposed target of Bcl11A in β globin cluster, we found that this lack of regulation is not linked to mutated Bcl 11a and nor to SNPs into its binding region. These results support the hypothesis that the control of Hb F is due to variation of the level of Bcl 11 A expression and the finding of a variation in the 5’UTR region which may interfere with the Bcl 11A RNA translation is another supporting evidence. Further studies of the consequence of this triplet insertion are ongoing to estimate its possible involvement in BCL11A transcription and translation and thus Bcl11A level.We confirmed that the “Sankaran region” is an important element of Bcl11A control of Hb F expression. A study of chromatin conformation (3C-qPCR) using patient bearing these small deletions are needed to test the hypothesis of a non-dissociation of the beta-globin LCR (locus control region) from its attachment point in the genes Gγ Aγ region confirming the major role of this site in the HbF downregulation. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-165: Low TCR diversity (divpenia) is a new prognosis factor of overall survival in metastatic breast cancer

Abstract Rationale Lymphopenia (&amp;lt;1000 Lymphocytes/μl) or CD4+ T cell lymphopenia (&amp;lt;450/μl), detected before initiation of chemotherapy are predictive factors for toxicity and death in metastatic solid tumors (Borg et al 2004; Ray Coquard et al 2009). The goal of the present study was to further identify the characteristics of the T cells in these lymphopenic patients. TCR diversity was investigated and tested as predictive factor for overall survival (OS). Patients and methods The ImmunTraCkeR® assay (ImmunID, Grenoble France), which analyzes, through semi quantitative multiplex PCR, the V-D-J combinatorial diversity of TCR-beta chain (TRB), was used to investigate diversity of T cell repertoire on cryopreserved blood samples from a prospective cohort of untreated metastatic breast cancer patients (SEMTOF study)(n=66). Univariate and multivariate analysis of prognostic factors for OS were investigated in this series as well as in a validation series. Results A severe T cell Divpenia (T cell diversity below 33%) (average diversity for healthy people is 70%) was associated with median survival of 9 months vs 24 months for the remaining patients (logrank p.value=0.0047). In a multivariate analysis, including haemoglobin level, polynuclear neutrophil count and liver metastasis, divpenia (defined at 30% threshold defined by ROC curve analysis) was identified as an independent prognostic factor. The NDL® score (Numeration Diversity Lymphocytes representation) that combines lymphocytes numeration with TRB diversity, demonstrated that lympho-divpenia (T cell diversity below 33% and lymphopenia at 1Giga/L threshold) was associated with a poor prognosis in term of patients survival compared to patients with either a good numeration or good diversity or both good numeration and diversity (p.value=0.0202). Prognostic value of NDL® score was then evaluated on a prospective validation cohort of 33 patients (logrank p.value=0.002). Conclusion: Divpenia, and a combined parameter NDL are predictive factors for survival in metastatic breast cancer patients. An open clinical study (LYMPHOS1) will investigate divpenia and NDL® score in a larger prospective cohort of metastatic breast patients. Acknowledgments M.MANUEL is a CIFRE recipient. This work (Proof of Concept “LYMPHOS1”) was financially supported by CLARA (LYON, France) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-165. doi:10.1158/1538-7445.AM2011-LB-165

Semi-quantitative PCR Analysis of DNA Degradation

Natural variables, including environmental conditions and post-mortem interval (PMI), affect the course of tissue decay in forensic and archaeological contexts. Compared with soft tissue, skeletal materials provide longer protection against DNA degradation. However environmental factors, including temperature and humidity, may affect the longevity of DNA survival in bone structure. In this study we describe the development of a semi-quantitative multiplex PCR based method to assess the level of DNA degradation with applications in analysis of forensic specimens. The assay was then applied to assess DNA survival in rib bone samples taken from 12 corpses in the mortuary. Samples were de-fleshed and stored in two different environmental conditions, including ambient conditions and underground burial from 103 to 445 days. Bone DNA was extracted and then assessed by the optimized semi-quantitative PCR and capillary electrophoresis of three products from the human mitochondrial DNA. The results show DNA survived for longer periods in samples stored in ambient conditions compared with underground burial conditions. There was no correlation found between the level of DNA degradation and PMI in our samples. Results also confirm that post-mortem alteration to DNA in skeletal tissues can occur very soon after death, with direct implications to the forensic industry.

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.

Copy Number Variation and Missense Mutations of the Agouti Signaling Protein (ASIP) Gene in Goat Breeds with Different Coat Colors

In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A^Wt (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A^Wt allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds.

Transcript accumulation of putative drought responsive genes in drought-stressed chickpea seedlings

Differential display reverse transcriptase PCR was used to identify cDNA sequences induced by drought in chickpea seedlings. The sequences of differentially expressed cDNAs: 192, 214, 219 and H1 showed high similarities at the protein level to known drought-inducible genes encoding for alanine aminotransferase, βAKIN1, a proteine kinase from the SnRK1 complex, lipid binding protein and COR protein, respectively. No significant similarity was found with the candidate sequence 277. Semi-quantitative multiplex PCR was used to verify that differentially amplified cDNAs were derived from differentially expressed genes. Relative quantification of the candidate sequences in the controls (well-watered seedlings) of drought-tolerant cv. ICCV2 and drought-susceptible cv. ILC3279 confirmed their induction upon drought in both cultivars. The transcript accumulation of the highly induced sequences, 219 and H1, was more important in the tolerant than the susceptible cultivar. These sequences are likely to be associated with drought tolerance in chickpea seedlings in contrast to sequences 214 and 277 which showed no variation in the mRNA accumulation between cultivars. The effect of ABA treatment on the mRNA accumulation of the isolated sequences 192, 214, 219 and H1 was analyzed. Sequences H1 and 192 were up-regulated by ABA treatment whereas sequences 214 and 219 were not, indicating an ABA-dependent and ABA-independent pathways in signal transduction in response to drought stress in chickpea seedlings. Key words: Differential display, DDRT-PCR, Cicer arietinum, drought stress.

A large X‐chromosomal deletion is associated with microphthalmia with linear skin defects (MLS) and amelogenesis imperfecta (XAI)

A female patient is described with clinical symptoms of both microphthalmia with linear skin defects (MLS or MIDAS) and dental enamel defects, having an appearance compatible with X-linked amelogenesis imperfecta (XAI). Genomic DNA was purified from the patient's blood and semiquantitative multiplex PCR revealed a deletion encompassing the amelogenin gene (AMELX). Because MLS is also localized to Xp22, genomic DNA was subjected to array comparative genomic hybridization, and a large heterozygous deletion was identified. Histopathology of one primary and one permanent molar tooth showed abnormalities in the dental enamel layer, and a third tooth had unusually high microhardness measurements, possibly due to its ultrastructural anomalies as seen by scanning electron microscopy. This is the first report of a patient with both of these rare conditions, and the first description of the phenotype resulting from a deletion encompassing the entire AMELX gene. More than 50 additional genes were monosomic in this patient.

SPANX-B and SPANX-C (Xq27 region) gene dosage analysis in Sicilian patients with melanoma

The incidence of melanoma has dramatically increased in many countries (it is 4.5 cases every 100 000 inhabitants in Sicily) and Xq27 region contains genes important in cancer like the SPANX (sperm protein associated with the nucleus in the X chromosome) gene family. These genes, made up of two exons separated by an intron of about 650 base pair, are expressed in sperm cells and in many tumours, including melanoma. These observations suggested that SPANX genes, or some of them, may be involved in melanoma development. The aim of this study was to investigate the genetic variability of SPANX-B and SPANX-C in a sample of Sicilian male population including patients with melanoma of the skin and controls. A total of 99 patients were enrolled in this study. They included: 17 male patients with cutaneous melanoma and 82 normal males. Semiquantitative fluorescent multiplex PCR dosage analysis was carried out to identify the variety of classes of SPANX-B and SPANX-C genes. Sixteen and 13 genetic classes were detected for SPANX-B and SPANX-C genes, respectively. A statistical significant difference for a particular class of SPANX-C gene was found comparing patients with melanoma and controls (P=0.011). Further investigations should be conducted to confirm these observations and to evaluate the possible implication of other genes of the region Xq27-28 in melanoma.