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Background The traditional exact method for inferring relationships between individuals from genetic data is not easily applicable in all situations that may be encountered in several fields of applied genetics. This study describes an approach that gives affordable results and is easily applicable; it is based on the probabilities that two individuals share 0, 1 or both alleles at a locus identical by state. Results We show that these probabilities (zi) depend on locus heterozygosity (H), and are scarcely affected by variation of the distribution of allele frequencies. This allows us to obtain empirical curves relating zi's to H for a series of common relationships, so that the likelihood ratio of a pair of relationships between any two individuals, given their genotypes at a locus, is a function of a single parameter, H. Application to large samples of mother-child and full-sib pairs shows that the statistical power of this method to infer the correct relationship is not much lower than the exact method. Analysis of a large database of STR data proves that locus heterozygosity does not vary significantly among Caucasian populations, apart from special cases, so that the likelihood ratio of the more common relationships between pairs of individuals may be obtained by looking at tabulated zi values. Conclusions A simple method is provided, which may be used by any scientist with the help of a calculator or a spreadsheet to compute the likelihood ratios of common alternative relationships between pairs of individuals.

The APC gene is a putative human tumor-suppressor gene responsible for adenomatous polyposis coli (APC), an inherited, autosomal dominant predisposition to colon cancer. It is also implicated in the development of sporadic colorectal tumors. The characterization of APC gene mutations in APC patients is clinically important because DNA-based tests can be applied for presymptomatic diagnosis once a specific mutation has been identified in a family. Moreover, the identification of the spectrum of APC gene mutations in patients is of great interest in the study of the biological properties of the APC gene product. We analyzed the entire coding region of the APC gene by the PCR–single-strand conformation polymorphism method in 42 unrelated Italian APC patients. Mutations were found in 12 cases. These consist of small (5–14 bp) base-pair deletions leading to frameshifts; all are localized within exon 15. Two of these deletions, a 5-bp deletion at position 3183–3187 and a 5-bp deletion at position 3926–3930, are present in 3/42 and 7/42 cases of our series, respectively, indicating the presence of mutational hot spots at these two sites.

Twenty-four sporadic colorectal adenomas were analysed for the presence of allelic loss on the short arm of chromosome 17 as well as mutations in the K-ras and p53 genes. Chromosome 17p13 allelic loss was not present in 14 out of 14 informative cases. K-ras mutations were observed in 15 out of 24 cases. A p53 gene mutation (GGC--GAC at codon 245) was detected in two biopsies taken at a four year interval from a recurrent rectal villous adenoma. Both biopsies also contained the same K-ras gene mutation (GGT--GTT at codon 12). The data from the recurrent rectal adenoma provide in vivo evidence that K-ras and p53 heterozygous mutations confer a proliferative advantage but together are not sufficient for malignant transformation.

The main objective of this pilot study was to assess the possibility of achieving engraftment of HLA-matched sibling donor mobilized hematopoietic stem cells after immunosuppressive non-myeloablative therapy. The second objective was to verify whether high-dose therapy with autologous stem cells rescue followed by allografting conditioned by only an immunosuppressive regimen, can be combined in order to achieve the reduction of tumor burden after autografting and the control of residual disease with immune-mediated effects after allografting.To enter the pilot study the patients had to fulfil the following criteria: advanced resistant disease, presence of an HLA matched sibling donor, no general contraindications to stem cell transplantation. Our data refers to 9 patients: Hodgkin's disease (n = 4), non-Hodgkin's lymphoma (n = 2), advanced chronic myelogenous leukemia (n = 2) (one patient with accelerated phase Ph-negative but p190 BCR-ABL gene positive by RT-PCR and one with Ph-positive blastic phase), refractory anemia with excess of blasts t(1;3) (p36;q21) (n = 1). All patients but one received the combined approach. At a median of 40 days (range 30-96), after high-dose therapy and autologous stem cell engraftment, the patients were treated with immunosuppressive therapy consisting of fludarabine and cyclophosphamide (Flu-Cy protocol) and then HLA matched donor mobilized stem cells were infused into the patients. GvHD prophylaxis consisted of cyclosporin and methotrexate.To date, with a median observation period of 4 months (range, 2-10), complete chimerism (100% donor cells) has been achieved in 6 patients. Three patients did not achieve complete chimerism: one patient died of progressive Hodgkin's disease when he reached 55% of donor cells, another patient is now in increasing phase of donor cell engraftment and the last patient (blastic phase-CML) was the only case who appears to have had autologous recovery. Two of the Hodgkin's disease patients, who were in partial remission after autografting, achieved complete remission after allografting and both are disease free 2 and 6 months after. Another Hodgkin's disease patient is alive at 10 months but she has progressive disease. One of the two patients with non-Hodgkin's lymphoma, who achieved partial remission after autografting, obtained complete remission and he is disease free 2 months after allografting. The other patient maintains partial remission obtained after autografting. The accelerated phase-CML patient obtained hematologic and molecular remission; the RAEB patient achieved hematologic and cytogenetic remission. In two patients severe aGVHD (grade II-III) was the single major complication but neither patient died of it. Mild aGVHD was seen in another patient. In only one patient did the ANC decrease to below 1 x 10(9)/L and in no case did platelets decrease below 20 x 10(9)/L. No patients required a sterile room or any red cell or platelet transfusions.Immunosuppressive therapy with a Flu-Cy protocol allowed engraftment of HLA-matched sibling donor stem cells without procedure-related deaths; moreover, we have demonstrated that this combined procedure can be pursued in safety in a serious ill population and some of these patients achieved a complete remission. This procedure is not likely to be curative, but a fascinating step along the path to curing these diseases. Of course, the follow-up is too short to document the incidence of cGvHD.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural'' and "urban'' samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes. (C) 2014 Elsevier Ireland Ltd. All rights reserved.