The Frequency of Germline BRCA and Non-BRCA HR-Gene-Variants in a Cohort of Pancreatic Cancer Patients.

Germline DNA alterations affecting homologous recombination pathway genes have been associated with pancreatic cancer (PC) risk. BRCA2 is the most studied gene and affects the management of PC patients and their families. Even though recent reports have suggested a similar role of germline ATM pathogenic variants (PV) in familial PC, there is still a disagreement between experts on how it could affect patient management given the lack of proper PC risk estimates. We retrospectively analyzed the germline data of 257 PC patients among whom nearly 50% were sporadic cases. We showed similar frequencies of BRCA2 (4.9%) and ATM (4.4%) PV or likely pathogenic variants, which were not related to familial history. Based on our findings and that of the literature, we suggest including ATM gene among the panel of genes analyzed in PC patients pending the publication of prospective studies.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

A novel homozygous MMP2 mutation in a family with Winchester syndrome.

The 2001 International Classification of Constitutional Disorders of Bone has included in the group of multicentric hands and feet osteolysis syndromes three autosomal recessive inherited disorders: Winchester, Torg and nodulosis-arthropathy-osteolysis (NAO) syndromes. Nosographic delineations of these rare syndromes are difficult to define, and there is no consensus. In 2001, two mutations in the matrix metalloproteinase 2 gene (MMP2) have been identified in two families with a NAO phenotype. In a recent study, a homozygous MMP2 mutation has also been identified in a patient presenting with Winchester syndrome. We report the clinical evolution of two sisters with a Winchester phenotype. Clinical review over 23 years provides information on the general evolution of osteolysis and points to an intrafamilial variation with clinical and radiological changes during the patients' life. In both sisters, we identified a new homozygous mutation in the catalytic domain of the MMP2 gene. Our study results are consistent with the involvement of MMP2 in Winchester syndrome and with the hypothesis that Winchester and NAO syndromes are allelic disorders that form a continuous clinical spectrum. At last, our observation emphasizes the interest of molecular analysis in genetic counselling of this consanguineous family.

Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells.

In our experience, patients with neuroblastoma who undergo transplantation with CD34+ cells following high-dose chemotherapy have prolonged delays in platelet recovery. In vitro expansion of megakaryocyte (MK) cells may provide a complementary transplant product able to enhance platelet production in the recipient. We investigated the ability of a combination of various hematopoietic growth factors to generate ex vivo MK progenitors. Immunoselected CD34+ cells from peripheral blood stems cells (PBSCs) were cultured in media with or without serum, supplemented by IL-3, IL-6, IL-11, SCF, TPO, Flt-3 ligand, and MIP-1alpha. In terms of MK phenotypes, we observed a maximal expansion of CD61+, CD41+, and CD42a of 69-, 60-, and 69-fold, respectively, i.e., 8-10 times greater than the expansion of total cell numbers. Whereas the absolute increment of CD34+ cells was slightly elevated (fourfold) we showed increases of 163-, 212-, and 128-fold for CD34+/CD61+, CD34+/CD41+, and CD34+/CD42a+ cells, respectively. We obtained only a modest expansion of CFU-MKs after only 4 days of culture (fourfold) and similar levels of CFU-MKs were observed after 7 days (fivefold). Morphology and immunohistochemistry CD41+ analyses confirmed expansion of a majority of CD41+ immature cells on days 4 and 7, while on day 10 mature cells began to appear. These results show that primarily MK progenitors are expanded after 4 days of culture, whereas MK precursor expansion occurs after 7 days. When we compared the two culture media (with and without serum) we observed that increases of all specific phenotypes of the MK lineage were more elevated in serum-free culture than in medium with serum. This difference was especially marked for CD34+/CD61+ and CD34+/CD41+ (163 vs 42 and 212 vs 36, respectively). We contaminated CD34+ cells with a neuroblastoma cell line and we observed no expansion of malignant cells in our culture conditions (RT-PCR for tyrosine hydroxylase positive at day 4 and negative at day 7). With our combination of hematopoietic growth factors we are able to sufficiently expand ex vivo MK late progenitor cells to be used as complementary transplant products in neuroblastoma patients who undergo transplantation with CD34+ cells. It is possible that these committed MK late progenitors could accelerate short-term platelet recovery in the recipient until more primitive progenitor cells have had time to engraft.

Large-volume leukapheresis procedure for peripheral blood progenitor cell collection in children weighing 15 kg or less: efficacy and safety evaluation.

BACKGROUND: We update our experience on large-volume leukapheresis (LVL) in very small patients with malignancies. LVLs were performed with the aim of reducing the psychological impact of leukaphereses by reducing the number of procedures while collecting large numbers of cells. PROCEDURE: Seventeen LVLs were performed using a Cobe Spectra separator in 14 patients weighing < or = 15 kg. A median of 3.8 patient's blood volumes corresponding to 296 mL/kg (range, 202-565) of blood was processed per session of 190 minutes (120-279) duration. A femoral catheter was installed specially for collection for 88% LVL (vs. 35% for standard leukaphereses). A median volume of 16.9 mL/kg was collected with 5.4 x 10(8) MNC/kg (range, 0.6-16.3) and 8.2 x 10(6) CD34+ cells/kg (range, 1.3-31.7). RESULTS: No signs of complications due to citrate toxicity were encountered. No hypotensive or hypothermic episodes were observed. Platelet counts were significantly diminished after each procedure (median: -59%). When the extracorporal line was not primed with red blood cells (RBC), the difference between pre-LVL and post-LVL hemoglobin levels was significant with a median 32 g/L decrease. CONCLUSIONS: The LVL approach for peripheral blood progenitor cells (PBPC) collection in very small children may expose them to the risk of anemia and thrombocytopenia and an excess of special central line installation. The application of this technique in these patients should be reserved for special cases when a very large number of cells must be collected and should be performed by an experienced team.

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts.

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.