Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise.

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.

Allogeneic hemopoietic SCT for patients with primary myelofibrosis: a predictive transplant score based on transfusion requirement, spleen size and donor type.

A total of 46 patients with primary myelofibrosis (PMF) (median age 51 years), underwent an allogeneic hemopoietic SCT (HSCT) after a thiotepa-based reduced-intensity conditioning regimen. The median follow-up for surviving patients is 3.8 years. In multivariate analysis, independent unfavorable factors for survival were RBC transfusions >20, a spleen size >22 cm and an alternative donor-24 patients had 0-1 unfavorable predictors (low risk) and 22 patients had 2 or more negative predictors (high risk). The overall actuarial 5-year survival of the 46 patients is 45%. The actuarial survival of low-risk and high-risk patients is, respectively, 77 and 8% (P<0.0001); this is because of a higher TRM for high-risk patients (RR, 6.0, P=0.006) and a higher relapse-related death (RR, 7.69; P=0.001). In multivariate Cox analysis, the score maintained its predictive value (P=0.0003), even after correcting for donor-patient age and gender, Dupriez score, IPSS (International Prognostic Scoring System) score pre-transplant and splenectomy. In conclusion, PMF patients undergoing an allogeneic HSCT may be scored according to the spleen size, transfusion history and donor type; this scoring system may be useful to discuss transplant strategies.

An unusual observation of tetragametic chimerism: forensic aspects.

A 41-year-old healthy Caucasian male showed an unidentifiable direct AB0 group and a B group by an indirect method revealing the presence of natural antibodies anti-A1 and anti-A2. Mixed fields with anti-B and anti-A+B antisera led to the conclusion that blood group B and 0 cell populations were present in a 1:1 ratio. A negative anamnesis for both transplantation and transfusion suggested a chimerism. DNA analysis of tissues revealed a tetragametic chimerism due to an apparent double parental contribution of nuclei in a phenotypically normal man.

Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): predictive role of minimal residual disease monitoring on relapse.

We developed a PCR-based method to monitor clonogenic IgH VDJ rearrangement as a possible predictor of relapse in patients with acute B-ALL after allogeneic bone marrow transplantation (BMT). We studied 23 patients at diagnosis, before and after BMT. At the time of BMT, 13 patients were in first complete remission, eight in second complete remission and two in relapse. Four patients were PCR negative before BMT and remained PCR negative also after BMT (-/- pattern). They are still in remission after a median follow-up of 41 months. Nineteen patients were MRD-positive before BMT: three were PCR negative at first determination after BMT (+/- pattern) and maintain remission. Sixteen patients were PCR-positive at first determination after BMT (+/+ pattern): five became PCR negative (+/+/- pattern) (four with chronic graft-versus-host disease (GVHD) and two after donor lymphocyte infusions (DLI)). Nine patients remained PCR-positive (+/+/+ pattern) (four remain in remission, and six relapsed); two patients died before transplant. In conclusion, PCR negative patients before BMT remained negative post-BMT; many pre-BMT positive patients had initial MRD positivity after BMT: 37% of them achieved a molecular remission with cGVHD or DLI.

Factors influencing haematological recovery after allogeneic haemopoietic stem cell transplants: graft-versus-host disease, donor type, cytomegalovirus infections and cell dose.

Platelet recovery after allogeneic haemopoietic stem cell transplant (HSCT) and predictive factors were analysed in 342 patients with haematological malignancies. All patients were prepared with cyclophosphamide plus total body irradiation, and received an unmanipulated HSCT from an HLA-identical sibling (n = 270), a matched unrelated donor (n = 67) or an identical twin (n = 5). The source of stem cells was peripheral blood (n = 15) or bone marrow (n = 327). Graft-vs.-host disease (GvHD) prophylaxis consisted of cyclosporin A with or without methotrexate. The proportion of patients with < 50 x 10(9)/l platelets on d +50, d +100, d +200 and d +365 after HSCT was 26%, 27%, 14% and 11% respectively. Thrombocytopenia was independent of the degree of complete donor chimaerism. Four variables were predictive of platelet recovery: donor type, acute GvHD, cytomegalovirus (CMV) infection and number of cells infused at transplant. Recipients of an unrelated graft had lower platelet counts (49 x 10(9)/l) on d +50 than identical sibling grafts (10(8) x 10(9)/l) (P < 0.001) and twin grafts (149 x 10(9)/l) (P < 0.001). Patients with GvHD grades 0, I, II, III and IV had significantly different platelet counts on d +50 (153 x 10(9)/l, 102 x 10(9)/l, 85 x 10(9)/l, 32 x 10(9)/l and 22 x 10(9)/l; P < 0.001) and thereafter. Thrombocytopenia was more frequent in patients with high-level CMV antigenaemia (> four positive cells/2 x 105) (P < 0.0001) and in patients who received a low cell dose at transplant (< or = 4.1 x 10(8)/kg) (P = 0.009). Platelet counts predicted transplant-related mortality (TRM) and were higher at all time intervals in patients surviving the transplant. Patients with grade II GvHD and > 50 x 10(9)/l platelets had a lower TRM than patients with grade II GvHD and < or = 50 x 10(9)/l platelets (14% vs. 40%, P < 0.0001). In conclusion, (i) a significant proportion of allogeneic HSCT recipients are thrombocytopenic long-term, irrespective of complete donor chimaerism, (ii) thrombocytopenia identifies patients at greater risk of lethal complications, and (iii) platelet recovery is influenced by GvHD, donor type, CMV infections and cell dose, not by stem cell source or other patient-disease-related variables.

Alternative donor transplants for patients with advanced hematologic malignancies, conditioned with thiotepa, cyclophosphamide and antithymocyte globulin.

Preparative regimens without total body irradiation (TBI) have been reported for alternative donor hemopoietic stem cell transplants (HSCT). Between 7 September 1994 and 7 June 1999 48 patients with advanced hematologic malignancies were conditioned with thiotepa (THIO) 15 mg/kg, cyclophosphamide (CY) 150 mg/kg and antithymocyte globulin (ATG). Donors were HLA mismatched family members (1-2 antigens) (FAM) (n = 24, median age 31 years) or HLA matched unrelated donors (UD) (n = 24, median age 34 years). GVHD prophylaxis was cyclosporine and methotrexate. Stem cell source was peripheral blood (n = 8) or bone marrow (n = 40). Hematologic recovery was seen in 42/46 (91%) evaluable patients and complete chimerism in 31/37 patients (85%). Acute GVHD grades III-IV were seen in 10/46 patients surviving 10 days (21%) and extensive chronic GVHD in 2/36 patients surviving 100 days (5%). Twenty-six patients died (54%), eight of recurrent disease (17%) and 18 of transplant-related complications (37%): main causes of TRM were GVHD (15%), infections (15%) and graft failure (4%). Twenty-two patients (46%) survive with a median follow-up of 877 days (287-1840). The actuarial 3-year survival is 49% for FAM and 42% for UD transplants. Results obtained with this regimen in unrelated grafts for advanced CML (n = 15) were not significantly different when compared to 21 concurrent UD grafts for advanced CML prepared with CY-TBI. In conclusion, the combination of THIO-CY-ATG allows engraftment of alternative donor hemopoietic stem cells. Results are similar when using unrelated matched donors or partially mismatched family donors, and not significantly different when compared to patients conditioned with CY-TBI.

Late graft failure 8 years after first bone marrow transplantation for severe acquired aplastic anemia.

A 14-year-old patient with acquired very severe aplastic anemia (VSAA) underwent bone marrow transplantation (BMT) from his HLA-identical brother. Preparative therapy was cyclophosphamide (CY) 200 mg/kg over 4 days. GVHD prophylaxis was with cyclosporin A (CsA) for a year. After an 8 year follow-up during which the patient was well with normal blood counts, graft failure occurred. At this time marrow chimerism studies demonstrated that 85% of hemopoiesis was of recipient origin. The patient was re-engrafted from the same donor after conditioning with CY 200 mg/kg over 4 days plus rabbit antithymocyte globulin (ATG) 3.5 mg/kg/day for 3 days. After 140 days follow-up he has a normal blood count. The possible causes of the graft failure are discussed. This case demonstrates that, although rarely, very late graft failure may occur after BMT for AA and highlights the need for long-term monitoring even in apparently successfully transplanted patients.

Engraftment of HLA-matched sibling hematopoietic stem cells after immunosuppressive conditioning regimen in patients with hematologic neoplasias.

BACKGROUND AND OBJECTIVE: 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. DESIGN AND METHODS: 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. RESULTS: 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. INTERPRETATION AND CONCLUSIONS: 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.

Evidence of cytogenetic and molecular remission by allogeneic cells after immunosuppressive therapy alone.

An immunosuppressive but not myeloablative regimen followed by HLA-matched donor mobilized haemopoietic stem cell transplantation was employed in two high-risk patients. The first patient had refractory anaemia with excess blasts (RAEB) and cytogenetic evidence of translocation 1;3(p36;q21). The second patient had Philadelphia-negative but p190 BCR-ABL chimaeric gene positive chronic myelogenous leukaemia in accelerated phase (AP-CML). The conditioning regimen consisted of fludarabine (30 mg/m2/d, days 1-3) with cyclophosphamide (300 mg/m2/d, days 1-3). Cyclosporine and methotrexate were employed for acute graft-versus-host disease (aGVHD) prophylaxis. In both cases the engraftment of donor cells was demonstrated by cytogenetics and short tandem repeat polymorphisms via PCR. Both patients are alive with normal cytogenetic (RAEB) and molecular (AP-CML) remissions, 100 and 150 d after allografting, respectively. In particular, in the AP-CML patient, the BCR-ABL became undetectable and the BCR-ABL/ABL ratio was <0.0001.

A 'de novo' point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia.

Severe hypercholesterolemia was found in an 11-year-old boy with no family history of familial hypercholesterolemia. The reduced LDL-receptor activity in cultured skin fibroblasts (40% 125I-LDL degradation as compared with a control cell line) indicated the presence of an LDL-receptor defect. The analysis of the promoter region and the exons of LDL-receptor gene by single strand conformation polymorphism revealed an abnormal migration pattern in exon 1, which was due to a T --> A transversion at nucleotide 28 of the cDNA. This novel mutation causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) and introduces an AviII restriction site in exon 1. Screening of the mutation by polymerase chain reaction (PCR) amplification of exon 1 and AviII digestion revealed that none of the proband's family members carried the mutation. Non-paternity was excluded after the analysis of a battery of 14 short tandem repeats located in 13 different chromosomes. These results are consistent with the hypothesis that the proband is heterozygous for a 'de novo' mutation of the LDL-receptor gene producing a non-conservative amino acid substitution. We suggest that the change in the net charge of the signal peptide, caused by the addition of a positively charged amino acid, impairs the co-translational translocation of the nascent receptor protein across the endoplasmic reticulum membrane.

Analysis of a short tandem repeat locus on chromosome 19 (D19S253).

A tetranucleotide tandem repeat locus on chromosome 19 (D19S253) was analysed. PCR products were detected by denaturing polyacrylamide gels with fluorescent-based technology. This study has confirmed a polymorphism with 9 alleles ranging from 209 to 241 bp with a simple repeat structure arranged from 7 to 15 repeats. Family studies confirmed mendelian inheritance of alleles. The efficiency on DNA extracted from bloodstains and cigarette butts has been evaluated. The protocol has shown sensitivity and reproducibility.

Forensic evaluation of HUMCD4: an Italian database.

The YTTTC pentanucleotide short tandem repeat polymorphism HumCD4 was studied in an Italian population sample. PCR products were compared to an allelic ladder by manual PAGE and silver staining. A total of 6 alleles ranging from 5 to 12 repeats were represented in the analysed sample, of which 3 alleles (10, 6 and 5 repeats) were predominant and displayed a combined frequency of 0.91. Successful amplification was obtained from different sources such as blood and urine stains, teeth and paraffin embedded tissues. Results were also determined in cases of severely degraded DNA. We consider that the HUMCD4 polymorphism may be a useful tool for individual identification, paternity testing, population studies and have also employed this locus to monitor engraftment of bone marrow transplantation.

HLA-DQA1 and amelogenin coamplification: a handy tool for identification.

A protocol for HLA-DQA1 and gender identification by single amplification is described. The use of the commercial HLA-DQA1 amplification kit (Perkin Elmer) permits a positive response for sex determination by adding primers for a short sequence on the first intron of Amelogenin gene. The suggested amplification protocol results in PCR products easily and clearly detectable on ethidium bromide stained agarose gel or silver stained polyacrylamide gel. In both gels the HLA-DQA1 observations at 242-239 bp are accomplished with a single band at 106 bp in females and a doublet 112-106 bp in males. HLA-DQA1 reverse dot-blot hybridization is unaffected by the presence of X and Y amplified fragments.

Individual identification of flood victims by DNA polymorphisms and autopsy findings.

On September 23rd 1993 Genova was flooded by heavy rainstorms and 4 people disappeared, including an elderly couple. Four days later a partially skeletonized body was found floating near the coast. No visual identification was possible. Autopsy findings were consistent with the medical history of a possible victim. DNA was extracted from a muscle sample and compared to paraffin embedded prostatic gland fragment taken by surgery. A positive identification could be made. On October 11th the body of a decomposed and partially skeletonized female was found. The visual identification was also uncertain and no clinical records were available. A blood sample from the son was obtained for maternal identification by the polymerase chain reaction.

A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient.

In this paper we report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In our patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3' breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement.

Genetic events in sporadic colorectal adenomas: K-ras and p53 heterozygous mutations are not sufficient for malignant progression.

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.

Identification of APC gene mutations in Italian adenomatous polyposis coli patients by PCR-SSCP analysis.

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.

Analysis of chimerism after bone marrow transplantation using specific oligonucleotide probes.

DNA hybridization with synthetic oligonucleotide probes was used to follow 18 leukemia patients who received bone marrow transplantation from HLA-identical siblings. Five oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed to produce simple restriction fragment length polymorphism patterns. Each probe hybridized to one or two bands in Hinf I-digested genomic DNA. Combined use of these probes enabled us to distinguish all sibling pairs. DNA analysis early post-transplant (15 days) detected donor-specific fragments in 14 of 18 subjects; two patients had a combination of recipient and donor fragments. Later post-transplant, (102-15 days), one of these two showed only recipient-specific fragments, and the other donor-specific fragments. These data are in accord with other markers of engraftment including cytogenetics and red blood cell phenotyping.