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.

The Italian National External quality assessment program in molecular genetic testing: results of the VII round (2010-2011).

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

Population data for 17 Y-chromosome STRs in a sample from Apulia (Southern Italy).

The 17 Y-STR loci included in the AmpFLSTR Yfiler PCR Amplification Kit were analyzed in 98 unrelated healthy males from Apulia (Southern Italy). A total of 97 different haplotypes were identified, of which 96 haplotypes were unique and 1 occurred twice. Allele frequencies for each Y-STR locus in pooled sample and estimated value of gene diversity (GD) were evaluated. The lowest value of GD was observed for DYS392 (0.126) and the highest one (0.936) for DYS385. The HD (haplotype diversity) for the studied Y-STR set showed a value of 0.9994, with an HMP (haplotype match probability) value of 0.0006, while the overall DC was 98.98%. Microvariant alleles were found for the DYS458 and DYS385 markers and sequenced. Furthermore, Φ(st)-based genetic distance computation and pair-wise analysis of molecular variance (AMOVA) test were carried out. When comparing our population with the Apulia sample previously investigated, the AMOVA analysis detected no evidence for significant differentiation. The comparison with all Italian populations submitted to the YHRD website showed no relevant differences with all Southern Italian populations (San Giorgio La Molara, Belvedere, Trapani and Catania) and significant genetic deviation with all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia). Moreover, the other populations and meta-populations belonging to the whole Mediterranean area (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) were different from our Apulia sample. The data were submitted to YHRD.

Allele frequencies of the new European Standard Set (ESS) loci in a population of Apulia (Southern Italy).

Allele frequencies of five miniSTRs loci (D1S1656, D2S441, D12S391, D10S1248 and D22S1045) included in the new European Standard Set (ESS) were calculated from a sample of 150 unrelated individuals from Apulia, a Region of Southern Italy. Two different PCR Amplification Kits were used, in order to evaluate the concordance of the genotypes. The results obtained with the two kits showed no differences in all genotype profiles. No deviation from Hardy-Weinberg expectations was detected at either locus. Moreover genetic analysis using Fst estimation showed no evidence for differentiation at the five new loci between Apulia and Italian populations. The high levels of polymorphisms of the analyzed markers in the Apulian population allow to confirm that these markers are useful tools in paternity and forensic analysis from degraded DNA samples.

The 2011 GeFI collaborative exercise. Concordance study, proficiency testing and Italian population data on the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045.

The 2011 collaborative exercise of the ISFG Italian Working Group GeFI was aimed at validating the five ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. The protocol required to type at least 50 multilocus profiles from locally resident individuals and two blind bloodstains in duplicate (i.e., using at least two different commercial kits), and to send the electropherograms to the Organizing Committee. Nineteen laboratories distributed across Italy participated, collecting a total of 960 samples. Full concordance was found for the five new miniSTRs as observed from the comparison of 13,150 alleles. The inspection of the electropherograms allowed the identification of a very limited number of mistypings in the miniSTR genotypes thus contributing to the establishment of an high quality Italian database of frequencies.

Novel splice isoforms of STRADalpha differentially affect LKB1 activity, complex assembly and subcellular localization.

STRADalpha is a pseudokinase that forms a heterotrimeric complex with the scaffolding protein MO25 and the tumor suppressor serine threonine protein kinase LKB1. Mutations in the LKB1 gene are responsible for the Peutz-Jeghers Syndrome (PJS) characterized by a predisposition to hamartomatous polyps and hyperpigmentation of the buccal mucosa. Mutations in LKB1 have also been observed in some sporadic tumours unrelated to PJS. The LKB1/STRAD/MO25 complex is involved in the regulation of numerous signaling pathways including metabolism, proliferation and cellular polarity of human intestinal epithelial cells. Cell polarization, together with tissue-restricted transcription, represents the main feature of enterocyte differentiation. Since a full-length STRADalpha transcript has not been identified thus far in these cells, we evaluated the expression of endogenous STRADalpha in five colorectal cancer cell lines characterized by their diverse ability to differentiate in vitro. We report herein the discovery of several novel splice isoforms of STRADalpha that differentially affect the kinase activity, complex assembly, subcellular localization of LKB1 and the activation of the LKB1-dependent AMPK pathway.

Cancer risks in LKB1 germline mutation carriers.

BACKGROUND AND AIMS: Germline mutations in the LKB1 gene are known to cause Peutz-Jeghers syndrome, which is an autosomal dominant disorder characterised by hamartomatous polyposis and mucocutaneous pigmentation. This syndrome is associated with an increased risk of malignancies in different organs but there is a lack of data on cancer range and risk in LKB1 germline mutation carriers. PATIENTS AND METHODS: The cumulative incidence of cancer in 149 Peutz-Jeghers syndrome patients with germline mutation(s) in LKB1 was estimated using Kaplan-Meier time to cancer onset analyses and compared between relevant subgroups with log rank tests. RESULTS: Thirty two cancers were found in LKB1 mutation carriers. Overall cancer risks at ages 30, 40, 50, 60, and 70 years were 6%, 18%, 31%, 41%, and 67%, respectively. There were similar overall cancer risks between male and female carriers. However, there were overall cancer risk differences for exon 6 mutation carriers versus non-exon 6 mutation carriers (log rank p=0.022 overall, 0.56 in males, 0.0000084 in females). Most (22/32) of the cancers occurred in the gastrointestinal tract, and the overall gastrointestinal cancer risks at ages 40, 50, 60, and 70 years were 12%, 24%, 34%, and 63%, respectively. In females, the risks for developing gynaecologic cancer at ages 40 and 50 years were 13% and 18%, respectively. CONCLUSIONS: Mutations in exon 6 of LKB1 are associated with a higher cancer risk than mutations within other regions of the gene. Moreover, this study provides age related cumulative risks of developing cancer in LKB1 mutation carriers that should be useful for developing a tailor made cancer surveillance protocol for Peutz-Jeghers syndrome patients.

Endoglin gene mutations and polymorphisms in Italian patients with hereditary haemorrhagic telangiectasia.

Autosomal-dominant hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous disease caused by mutations in at least two different loci. We screened for mutations in four Italian families where segregation studies showed clear evidence of linkage to the endoglin (ENG) locus. In addition, one sporadic case and three patients with pulmonary arteriovenous malformations, belonging to small nuclear families unsuitable for linkage analysis, were included in the screening. The proband from each family was investigated using single-strand conformation polymorphism and heteroduplex analysis; potential variants were sequenced. Four novel and one previously reported mutation were detected, as well as three new polymorphisms. The novel mutations included deletions in exon 1 (patient 581/02), exon 5 (patient 780/01) and exon 7 (patient 700/01), and a C-->T229 substitution in exon 3 (patient 462/02). When analysing patient 700/01 and his affected daughter, we encountered a mutant ENG allele with two mutations--a deletion in exon 7 and a substitution in exon 12--which converts isoleucine 575 into threonine, in a non-conserved region. Both mutations were absent in the two healthy sons of the patient, while the polymorphic variant in exon 12 was present in his healthy father. These results and haplotype-segregation studies suggest that a de novo deletion had occurred in the gamete of paternal origin. For the first time the parental germline in which a de novo HHT mutation occurred has been identified.

Functional analysis of LKB1/STK11 mutants and two aberrant isoforms found in Peutz-Jeghers Syndrome patients.

Peutz-Jeghers Syndrome (PJS) is thought to be caused by mutations occurring in the widely expressed serine/threonine protein kinase named LKB1/STK11. Recent work has led to the identification of four mutants (R304W, I177N, K175-D176del, L263fsX286) and two novel aberrant LKB1/STK11 cDNA isoforms (r291-464del, r485-1283del) in a group of PJS Italian patients. Three of the four mutations only change 1 or 2 amino acids in the LKB1/STK11 catalytic domain. Here we demonstrate that all six LKB1/STK11 variants analysed are completely inactive in vitro as they were unable to autophosphorylate at Thr336, the major LKB1/STK11 autophosphorylation site, and to phosphorylate the p53 tumour suppressor protein. We also show that 5 out of the 6 variants are entirely localised in the nucleus in contrast to the wild type LKB1/STK11, which is detected in both the nucleus and cytoplasm. Finally we demonstrate that all 6 LKB1/STK11 variants, in contrast to wild type LKB1/STK11, are unable to suppress the growth of melanoma G361 cells. Taken together, these results demonstrate that the LKB1 mutations investigated in this study lead to the loss of serine/threonine kinase activity and are therefore likely to be the primary cause of PJS development in the patients that they were isolated from.

Two novel mutations and a new STK11/LKB1 gene isoform in Peutz-Jeghers patients.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disorder with variable expression and incomplete penetrance characterized by mucocutaneous pigmentation, predisposition to hamartomatous intestinal polyposis, and various other neoplasms. It occurs in approximately 1 in 8,300 to 29,000 live births. In nearly 50% of patients PJS is caused by germ line mutations in the STK11/LKB1 serine/threonine kinase gene, the only kinase gene currently known to act as a tumor suppressor. We have performed a mutation search in the STK11/LKB1 gene in 8 sporadic cases and 3 PJS families using a combination of different screening techniques. We have identified four mutations, two of which I177N and the IVS2+1A->G, were previously unreported. We have also evaluated the presence of cDNA alterations by means of RT-PCR analysis and direct cDNA sequencing and have found two aberrant transcripts in a single PJS case despite the lack of any apparent genomic alteration. Finally, we report the presence of a novel STK11/LKB1 cDNA isoform observed in all the normal subjects studied as well as in the majority of the PJS patients.

Cyclin E and chromosome instability in colorectal cancer cell lines.

AIMS/BACKGROUND: The development of colorectal cancer depends on at least two distinct pathways involving genetic instability, namely: chromosome instability (CIN) and microsatellite instability. Cyclin E is involved in aneuploidy and several cancer types show an abnormal number of chromosomes. METHODS: Cyclin E protein and mRNA values were analysed in human fetal skin fibroblasts and five colorectal cancer cell lines. RESULTS: Cells with an aberrant number of chromosomes had higher cyclin E mRNA values and a significant increase in protein concentrations. CONCLUSIONS: These data suggest that cyclin E regulation is altered in aneuploid cells and is an important factor in the CIN pathway.

Y-chromosome haplotypes in Italy: the GEFI collaborative database.

A sample of 1176 males from 10 Italian regions have been typed for DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, and DYS385. Individual haplotype data are available on line. A low degree of variation is present among regions. Use of this database is specifically recommended for forensic applications in Italy.

The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides.

Bombesin-like peptides (BLP) and their receptors are widely distributed throughout the intestine and are potential mitogens for gastrointestinal cancers. In this study we characterized the proliferation induced by BLP in the human adenocarcinoma cell line HT-29. The number of HT-29 cells, partially serum deprived (1% fetal bovine serum) for 48 h, was increased after 24 h of stimulation with bombesin, GRP, neuromedin B (NMB) and neuromedin C (NMC) ranging from 0.1 nM up to 1 microM. Reverse transcription polymerase chain reaction studies, revealed the presence of mRNA for NMB and for the GRP preferring receptor (GRP-R). mRNA for GRP, NMB preferring receptor (NMB-R) and bombesin receptor subtype 3 (BRS-3) were not detected. [D-Phe(6)]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2), are considered as inhibitors of binding to GRP-R and NMB-R, respectively. Surprisingly, A1 and A2 stimulated the proliferation of HT-29 cells. Moreover, in the simultaneous presence of 1 microM A1 and 0.1 microM GRP or 0.1 nM or 0.1 microM bombesin, inhibition of the proliferation was observed. Our data demonstrate that the proliferation induced by BLP in HT-29 cells is due to interaction with the GRP-R.

Nine novel APC mutations in Italian FAP patients.

Familial adenomatous polyposis (FAP) is a common hereditary syndrome characterized by early development of colorectal cancer consequent to extensive adenomatous polyps of the colon. In addition to the colonic manifestations the syndrome presents several extracolonic features including polyps of the upper gastrointestinal tract, congenital hypertrophy of the retinal pigment, jaw cysts, osteomata and desmoid tumors. In this study the entire APC coding region has been analysed for mutation in a panel of one Turcot and 33 unrelated Italian FAP patients using SSCP analysis, PTT and DNA sequencing. We detected APC mutations in 23 of them and identified nine which, to our knowledge were not previously reported. All of these novel mutations are in exon 15, including two nonsense mutations, 6 deletions or insertions leading to premature termination of the protein and one missense mutation (7697G>A). This last mutation occurs in the EB1-binding domain of the APC protein and segregates in four relatives of the patient with three of them presenting 2-3 adenomatous polyps.