The BRCA1 c.4096+1G>A Is a Founder Variant Which Originated in Ancient Times.

Approximately 30-50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45-79 percent and 39-48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago-around 3000 years ago.

Fleece variation in alpaca (Vicugna pacos): a two-locus model for the Suri/Huacaya phenotype.

BACKGROUND: Genetic improvement of fibre-producing animal species has often induced transition from double coated to single coated fleece, accompanied by dramatic changes in skin follicles and hair composition, likely implying variation at multiple loci. Huacaya, the more common fleece phenotype in alpaca (Vicugna pacos), is characterized by a thick dense coat growing perpendicularly from the body, whereas the alternative rare and more prized single-coated Suri phenotype is distinguished by long silky fibre that grows parallel to the body and hangs in separate, distinctive pencil locks. A single-locus genetic model has been proposed for the Suri-Huacaya phenotype, where Huacaya is recessive. RESULTS: Two reciprocal experimental test-crosses (Suri x Huacaya) were carried out, involving a total of 17 unrelated males and 149 unrelated females. An additional dataset of 587 offspring of Suri x Suri crosses was analyzed. Segregation ratios, population genotype frequencies, and/or recombination fraction under different genetic models were estimated by maximum likelihood. The single locus model for the Suri/Huacaya phenotype was rejected. In addition, we present two unexpected observations: 1) a large proportion (about 3/4) of the Suri animals are segregating (with at least one Huacaya offspring), even in breeding conditions where the Huacaya trait would have been almost eliminated; 2) a model with two different values of the segregation ratio fit the data significantly better than a model with a single parameter. CONCLUSIONS: The data support a genetic model in which two linked loci must simultaneously be homozygous for recessive alleles in order to produce the Huacaya phenotype. The estimated recombination rate between these loci was 0.099 (95% C.L. = 0.029-0.204). Our genetic analysis may be useful for other species whose breeding system produces mainly half-sib families.

HYPNOTIZABILITY-RELATED FAAH C385A POLYMORPHISM: POSSIBLE ENDOCANNABINOID CONTRIBUTION TO SUGGESTION-INDUCED ANALGESIA.

Fatty acid amide hydrolase (FAAH) degrades the endogenous endocannabinoid (eCB) anandamide and might be involved in the response to suggestions of analgesia in subjects with high hypnotizability scores (highs). Since the A allele of the FAAH C385A polymorphism (rs324420) is associated with lower FAAH activity, it was studied in 21 highs, 66 low hypnotizable individuals (lows), and 172 individuals not selected for hypnotizability (controls) representing the general population. No significant difference was observed among groups, but the A allele frequency showed a significant trend to increase from lows to controls and from controls to highs. Since eCB small differences can be amplified by eCB interactions with other neurotransmitters, a contribution of the FAAH polymorphism to the highs' analgesia should not be excluded.

Disaster victim identification by kinship analysis: the Lampedusa October 3rd, 2013 shipwreck.

On October 3rd, 2013 a boat carrying more than 500 migrants coming mostly from the Horn of Africa (Eritrea, Somalia, Ethiopia) sank near Lampedusa, a small Italian Island in the middle of the Mediterranean Sea. The recovered bodies were examined by a forensic team, and post mortem data (anthropological and odontological records, and DNA) were collected for identification. Genetic profiles based on 16 autosomal STRs were acquired from both victims and putative relatives recruited following an international call. The final genetic database included 363 victims and 43 reference persons from 36 independent families recruited until mid-2017, who were missing 35 first-degree and 6 second-degree relatives. A pairwise blind search approach was used to identify familial relationships within the victims and between victims and putative relatives. Two statistics were calculated, the Identity by State (IBS) and the Identity by Descent (IBD), the latter by using the DVI module of the FAMILIAS3 software to compute LRs and posterior probabilities. The putative identifications were confirmed in pedigree analysis using the information provided by the relatives. In selected cases, additional autosomal and lineage (Y-chromosome and mtDNA) markers were typed. Some critical points were highlighted: the lack for accurate allele and haplotype frequencies in African populations, especially for the lineage markers, and the need for a shared approach to the biostatistical interpretation of the results in DVI. In the end, 29 first-degree (parent-child and full sibs) out of 35 missing (83%), and 3 out of 6 of second-degree relatives (50%) showed a high statistical confidence for a positive identification. This study represents the first attempt to systematically deal with the genetic identification of African migrants who died in the Mediterranean Sea. The methodological and statistical approach used in this study was proved to be reliable and appropriate for future genetic identifications in other similar mass disasters.

Haplotype analysis of the H63D, IVS2+4t/c, and C282Y polymorphisms of the HFE gene reveals rare events of intragenic recombination.

OBJECTIVE: Two missense mutations of the HFE gene, one (C282Y) being a major gene for hereditary hemochromatosis and the other (H63D) playing a minor role in this disease, are carried by different haplotypes. Among other sequence variants of HFE, IVS2+4t/c polymorphism has been reported as a possible splicing mutation or risk modifier. Our aims were to identify sequence variants possibly associated with iron overload in our population, to study the intragenic haplotypes of the HFE gene, and to evaluate the role of IVS2+4t/c in hyperferritinemia. METHODS: We screened by direct sequencing the coding sequence and intron-exon boundaries of HFE in 265 patients with hyperferritinemia and 185 subjects from the general population. RESULTS: Linkage disequilibrium between the three pairs of polymorphic sites was complete between H63D and C282Y, whereas all four gametic types were present for both the H63D-IVS2+4t/c and the IVS2+4t/c-C282Y site pairs. The data supported a model in which the IVS2+4t/c polymorphism was ancestral, the D(63) mutation occurred on the t chromosome, and the Y(282) mutation occurred on the c chromosome; after the population spread of both mutations, intragenic recombination occurred on both sides of the t/c polymorphism, generating the rare haplotypes D(63)-c(IVS2+4)-C(282) and H(63)-t(IVS2+4)-Y(282). CONCLUSIONS: The IVS2+4c/t is a neutral polymorphism with regard to risk of iron overload. The presence of recombinant haplotypes on both its sides suggests a considerable evolutionary age of the two main risk alleles.

Polymorphism of Opioid Receptors µ1 in Highly Hypnotizable Subjects.

The possible cooperation between hypnotizability-related and placebo mechanisms in pain modulation has not been consistently assessed. Here, we investigate possible genetic bases for such cooperation. The OPRM1 gene, which encodes the µ1 opioid receptor-the primary site of action for endogenous and exogenous opioids-is polymorphic in the general population for the missense mutation Asn40Asp (A118G, rs1799971). The minor allele 118G results in decreased levels of OPRM1 mRNA and protein. As a consequence, G carriers are less responsive to opioids. The aim of the study was to investigate whether hypnotizability is associated with the presence of the OPRM1 polymorphism. Forty-three high and 60 low hypnotizable individuals, as well as 162 controls, were genotyped for the A118G polymorphism of OPRM1. The frequency of the G allele was significantly higher in highs compared to both lows and controls. Findings suggest that an inefficient opioid system may be a distinctive characteristic of highs and that hypnotic assessment may predict lower responsiveness to opioids.

Challenges in the identification of dead migrants in the Mediterranean: The case study of the Lampedusa shipwreck of October 3rd 2013.

Every year thousands of migrants die during the endeavour to reach the Italian coasts, making the Mediterranean the theatre of one of the greatest tragedies of mankind. Over 60% of these victims is buried unidentified: one of the reasons behind this is related to the specific difficulties and lack of strategies concerning AM and PM data collection. The present article describes how Italy is trying to face the problem of migrant identification, thanks to the collaboration between government, the Italian national police and universities. In particular, this is the first pilot study carried out to identify the victims of the second greatest tragedy of its kind off the Italian coast, near Lampedusa, on October 3rd 2013, which caused 366 victims. The present article shows the strategies conceived to collect postmortem and especially antemortem data and to compare them to identify matches, using medicolegal, anthropological, odontological and genetic approaches. Thirty-one victims out of 53 missing sought by relatives were identified (58.5%). The type and the quality of antemortem data available, generally photos and videos, pinpoints the importance of the face and the body for identification when the bodies are well preserved and how DNA analyses may at times present difficulties. In fact, critical points emerged concerning especially the lack of genetic information of the populations to which the victims belonged, the number of genetic markers needed to reach a statistical support for the identification and the need to adopt lineage markers such as mitochondrial DNA and Y-chromosome polymorphisms to identify parental relationships. This pilot study however has proven that families continue to seek their relatives and that it is possible, as well as mandatory, to identify migrant victims in spite of the difficulties in the collection of antemortem and postmortem data. In addition, considering the peculiar scenario, novel strategies for positive identification have to be defined in each field (anthropological, odontological and genetic) as well as in combination.

Cancer risk among the relatives of patients with pancreatic ductal adenocarcinoma.

BACKGROUND/AIMS: Pancreatic cancer is a leading cause of cancer-related death; the most consistently identified risk factors are smoking and family history. Our aims were to examine familial aggregations of pancreas and other cancers, and to determine the relative risk of the family members. METHODS: We prospectively collected data on the families of patients presenting with pancreatic ductal adenocarcinoma. Smoking habits and alcohol consumption of the probands were compared with the available statistics on the Italian population. Mortality from cancer was investigated in first-degree relatives, and age-dependent risks of dying from pancreatic cancer and other tumors were compared with background population levels. RESULTS: Data for 570 families were collected, including 9,204 relatives. Probands were 3- to 5-fold more often heavy smokers than the general population, and 9.3% of them reported a positive family history of pancreatic cancer. In first-degree relatives, only mortality from pancreatic cancer was significantly increased (relative risk at age 85 years = 2.7). Lifetime risk of dying of pancreas cancer was 4.1% for the relatives of all probands, and was 7.2% for the relatives of probands who developed disease before 60 years of age. CONCLUSIONS: The data suggest that genetic susceptibility to pancreatic cancer may be attributable, in addition to BRCA2, to moderate- to low-penetrance gene(s).

Validation of a large Italian Database of 15 STR loci.

Results from a collaborative exercise with proficiency testing conducted by 20 Italian laboratories on the 15 loci included in the Identifiler kit were analyzed by allele sharing methods and by standard population genetics tests. The validated database, including about 1500 subjects, was merged with that of a previous exercise conducted on nine loci, and the resulting allele frequencies, subdivided by Italian region, were published on-line.

Haplotypic structure of the X chromosome in the COGA population sample and the quality of its reconstruction by extant software packages.

BACKGROUND: The haplotypes of the X chromosome are accessible to direct count in males, whereas the diplotypes of the females may be inferred knowing the haplotype of their sons or fathers. Here, we investigated: 1) the possible large-scale haplotypic structure of the X chromosome in a Caucasian population sample, given the single-nucleotide polymorphism (SNP) maps and genotypes provided by Illumina and Affimetrix for Genetic Analysis Workshop 14, and, 2) the performances of widely used programs in reconstructing haplotypes from population genotypic data, given their known distribution in a sample of unrelated individuals. RESULTS: All possible unrelated mother-son pairs of Caucasian ancestry (N = 104) were selected from the 143 families of the Collaborative Study on the Genetics of Alcoholism pedigree files, and the diplotypes of the mothers were inferred from the X chromosomes of their sons. The marker set included 313 SNPs at an average density of 0.47 Mb. Linkage disequilibrium between pairs of markers was computed by the parameter D', whereas for measuring multilocus disequilibrium, we developed here an index called D*, and applied it to all possible sliding windows of 5 markers each. Results showed a complex pattern of haplotypic structure, with regions of low linkage disequilibrium separated by regions of high values of D*. The following programs were evaluated for their accuracy in inferring population haplotype frequencies: 1) ARLEQUIN 2.001; 2) PHASE 2.1.1; 3) SNPHAP 1.1; 4) HAPLOBLOCK 1.2; 5) HAPLOTYPER 1.0. Performances were evaluated by Pearson correlation (r) coefficient between the true and the inferred distribution of haplotype frequencies. CONCLUSION: The SNP haplotypic structure of the X chromosome is complex, with regions of high haplotype conservation interspersed among regions of higher haplotype diversity. All the tested programs were accurate (r = 1) in reconstructing the distribution of haplotype frequencies in case of high D* values. However, only the program PHASE realized a high correlation coefficient (r > 0.7) in conditions of low linkage disequilibrium.

Development of a heptaplex PCR system to analyse X-chromosome STR loci from five Italian population samples. A collaborative study.

Many X-chromosome short tandem repeats (X-STRs) have been validated for forensic use even if further studies are needed on allele frequencies and mutation rates to evaluate the extent of polymorphism in different populations and to establish reference databases useful for forensic applications and for anthropological studies. A single multiplex reaction of seven X-STRs, which includes the DXS6789, HUMARA, DXS10011, DXS7423, HPRTB, DXS6807, DXS101 loci, is presented and their allele frequency distribution in a large population sample including 556 subjects (268 females and 288 males) analysed by five forensic laboratories of Central and Northern Italy is shown. Our results demonstrate the feasibility of a single amplification/detection reaction involving seven markers of the X chromosome, which can be fruitfully used in complex kinship analysis.

False deletion of the D15S986 maternal allele in a suspected case of Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a neurological disorder caused by genetic defects of the chromosome region 15q11-q13; some 70-80% of cases are due to deletions of the maternal allele, as the paternal copy is imprinted. DESIGN AND METHODS: A maternal deletion at D15S986 was reported in a suspected case of AS; this marker is located in intron 2 of the ATP10C gene, which has been implicated in the development of AS. A segment of ~830bp, including this marker and the primers used in routine genetic test, was cloned and sequenced. RESULTS: A single nucleotide deletion (named ATP10C*c.760+3808delA, GenBank accession number HQ856823) was detected in the middle of the forward primer, leading to allele dropout. A large European population sample (N=363) was typed, and the detected variant was characterized as a novel polymorphism, with allele frequencies of 0.882 (TAT allele) and 0.118 (T-T allele). CONCLUSIONS: An alternative primer set was developed, for which the segregation pattern of D15S986 in the proband extended family was normal. It can replace the currently used set.

Haplotype analysis of BRCA1 gene reveals a new gene rearrangement: characterization of a 19.9 KBP deletion.

Germ-line mutations in the BRCA1 gene cause hereditary predisposition to breast and ovarian cancer. BRCA1 and BRCA2 mutations account for about 40% of high-risk families. Mutation-screening methods generally focus on genomic DNA and are usually PCR based; they enable the detection of sequence alterations such as point mutations and small deletions and insertions. However, they do not allow the detection of partial or entire exon(s) loss, because the presence of the homologous allele results in a positive PCR signal, giving rise to a false-negative result. Identification of unusual haplotypes in patient samples by an expectation maximization algorithm has recently been suggested as a method for identifying hemizygous regions caused by large intragenic deletions. Using a similar approach, we identified a novel BRCA1 genomic rearrangement in a breast/ovarian cancer family negative at the first mutation screening; we detected a deletion encompassing exons 14-19, probably due to replication slippage between Alu sequences.

Penetrances of breast and ovarian cancer in a large series of families tested for BRCA1/2 mutations.

Accurate estimates of breast and ovarian cancer penetrance in BRCA1/2 mutation carriers are crucial in genetic counseling. Estimation is difficult because of the low frequency of mutated alleles and the often-uncertain mechanisms of family ascertainment. We estimated the penetrances of breast and ovarian cancers in carriers of BRCA1/2 mutations by maximizing the retrospective likelihood of the genetic model, given the observed test results, in 568 Italian families screened for germline mutations. The software BRCAPRO was used as a probability calculation tool in a Markov Chain Monte Carlo approach. Breast cancer penetrances were 27% (95% CI 20-34%) at age 50 years and 39% (27-52%) at age 70 in BRCA1 carriers, and 26% (0.18-0.34%) at age 50 and 44% (29-58%) at age 70 in BRCA2 carriers, and ovarian cancer penetrances were 14% (7-22%) at age 50 and 43% (21-66%) at age 70 in BRCA1 carriers and 3% (0-7%) at age 50 and 15% (4-26%) at age 70 in BRCA2 carriers. The new model gave a better fit than the current default in BRCAPRO, the likelihood being 70 log units greater; in addition, the observed numbers of mutations in families stratified by gene and by cancer profile were not significantly different from those expected. Our new penetrance functions are appropriate for predicting breast cancer risk, and for determining the probability of carrying BRCA1/2 mutations, in people who are presently referred to genetic counseling in Italy. Our approach could lead to country-customized versions of the BRCAPRO software by providing appropriate population-specific estimates.

Inferring relationships between pairs of individuals from locus heterozygosities.

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.

Population data of four X-chromosome markers in Tuscany, and their use in a deficiency paternity case.

Four X-chromosome markers (DXS101, HPRTB, DXS8377, and STRX-1) were typed in a population sample from Tuscany, Italy, using a single amplification reaction. Resolution of a deficiency paternity case involving two women (either they were half-sisters or non-relatives) was made possible by typing these marker in addition to 16 conventional autosomal markers.

Allele sharing in first-degree and unrelated pairs of individuals in the Ge F I AmpFlSTR Profiler Plus database.

Eleven Italian forensic laboratories participated in a population study based on the AB Profiler Plus loci with proficiency testing. The validated database, including 1340 individuals, is available on-line. Tests for Hardy-Weinberg equilibrium, gametic unbalance, and heterogeneity of gene frequency were generally not significant. Gene frequencies at each locus were consistent with those of two previously published Italian studies, but different from a third. Individuals of each subsample were paired, and the total number of alleles shared across the nine loci was determined in each pair. The analysis was replicated over the total sample. In addition, two samples of mother-child pairs (N=315) and full-sib pairs (N=91) were subjected to allele sharing analysis. The resulting distributions were sufficiently distinct from the sample of unrelated pairs as to be of practical usefulness.

Hypnotizability and Catechol-O-Methyltransferase (COMT) polymorphysms in Italians.

Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT) in subjects with high hypnotizability scores (highs) has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotizability and the COMT single nucleotide polymorphism (SNP) rs4680 (Val(158)Met) were inconsistent. Here, we used a selective genotyping approach to re-evaluate the association between hypnotizability and COMT in the context of a two-SNP haplotype analysis, considering not only the Val(158)Met polymorphism, but also the closely located rs4818 SNP. An Italian sample of 53 highs, 49 low hypnotizable subjects (lows), and 57 controls, were genotyped for a segment of 805 bp of the COMT gene, including Val(158)Met and the closely located rs4818 SNP. Our selective genotyping approach had 97.1% power to detect the previously reported strongest association at the significance level of 5%. We found no evidence of association at the SNP, haplotype, and diplotype levels. Thus, our results challenge the dopamine-based theory of hypnosis and indirectly support recent neuropsychological and neurophysiological findings reporting the lack of any association between hypnotizability and focused attention abilities.

Estimated prevalence of canine Type 2 Von Willebrand disease in the Deutsch-Drahthaar (German Wirehaired Pointer) in Europe.

Type 2 Von Willebrand disease (VWD) is a severe coagulopathy occurring in the Deutsch-Drahthaar dog (or German Wirehaired Pointer, DD/GWP). Recently, a causative recessive mutation has been identified, and a DNA test is now available for individual screening. The genotype distribution (clear, carrier, and affected dogs) was investigated in 1855 DD/GWP dogs using data collected by the DD DNA-VWD-Databank in several European countries. 1704 (91.8%) DD/GWP dogs were genotypically clear of the VWD mutation, 144 (7.8%) were carriers, and seven (0.4%) were affected. The estimated disease allele frequency was highest in Germany and Sweden (almost 5%), and about 1% in Denmark, Finland and Norway. The Hardy-Weinberg equilibrium was tested in the German sample, and showed no evidence of deviation.