Microscopic Evidence of Malaria Infection in Visceral Tissue from Medici Family, Italy.

Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.

The Lady from Basel's Barfüsserkirche - Molecular confirmation of the Mummy's identity through mitochondrial DNA of living relatives spanning 22 generations.

The identity of the mummified Lady from the Barfüsser Church in Basel, Switzerland has been unsolved for decades, despite the prominent location of the burial place in front of the choir screen. A recent multidisciplinary research approach came up with a possible candidate, Anna Catharina Bischoff who died in Basel in 1787 with an age of 69 years (1719-1787). To verify the identity of the mummy, genealogists of the Citizen Science Basel discovered three living individuals of the maternal lineage of two different family branches, separated from Anna Catharina Bischoff by up to 22 generations. In this study we compare the ancient mitochondrial DNA of the mummy recovered from a premolar to the mitochondrial DNA of these three candidates. Initially the mitochondrial hypervariable regions I and II of the living individuals were screened using the Sanger sequencing method. This was followed by a mitochondrial capture approach and next generation sequencing to enrich for the whole mitochondrial genome of the mummy and one living person. A full mitochondrial genome has been recovered of both individuals sharing an identical haplotype. The sequence was assigned to the mitochondrial haplogroup U5a1+!16192 including two private mutations 10006G and 16293C. Only by using an interdisciplinary approach combining ancient DNA analysis and genealogy a maternal lineage of a non-noble family spanning 22 generations could be confirmed.

The Iceman's Last Meal Consisted of Fat, Wild Meat, and Cereals.

The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [1], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-year-old European glacier mummy [2, 3]. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual.

The 5300-year-old Helicobacter pylori genome of the Iceman.

The stomach bacterium Helicobacter pylori is one of the most prevalent human pathogens. It has dispersed globally with its human host, resulting in a distinct phylogeographic pattern that can be used to reconstruct both recent and ancient human migrations. The extant European population of H. pylori is known to be a hybrid between Asian and African bacteria, but there exist different hypotheses about when and where the hybridization took place, reflecting the complex demographic history of Europeans. Here, we present a 5300-year-old H. pylori genome from a European Copper Age glacier mummy. The "Iceman" H. pylori is a nearly pure representative of the bacterial population of Asian origin that existed in Europe before hybridization, suggesting that the African population arrived in Europe within the past few thousand years.

Metagenomic analysis reveals presence of Treponema denticola in a tissue biopsy of the Iceman.

Ancient hominoid genome studies can be regarded by definition as metagenomic analyses since they represent a mixture of both hominoid and microbial sequences in an environment. Here, we report the molecular detection of the oral spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a 5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA) specific reads. Through ranking the reads by abundance a relatively high number of rRNA reads most similar to T. denticola was detected. Mapping of the metagenome sequences against the T. denticola genome revealed additional reads most similar to this opportunistic pathogen. The DNA damage pattern of specifically mapped reads suggests an ancient origin of these sequences. The haematogenous spread of bacteria of the oral microbiome often reported in the recent literature could already explain the presence of metagenomic reads specific for T. denticola in the Iceman's bone biopsy. We extended, however, our survey to an Iceman gingival tissue sample and a mouth swab sample and could thereby detect T. denticola and Porphyrimonas gingivalis, another important member of the human commensal oral microflora. Taken together, this study clearly underlines the opportunity to detect disease-associated microorganisms when applying metagenomics-enabled approaches on datasets of ancient human remains.

Demographic histories, isolation and social factors as determinants of the genetic structure of Alpine linguistic groups.

Great European mountain ranges have acted as barriers to gene flow for resident populations since prehistory and have offered a place for the settlement of small, and sometimes culturally diverse, communities. Therefore, the human groups that have settled in these areas are worth exploring as an important potential source of diversity in the genetic structure of European populations. In this study, we present new high resolution data concerning Y chromosomal variation in three distinct Alpine ethno-linguistic groups, Italian, Ladin and German. Combining unpublished and literature data on Y chromosome and mitochondrial variation, we were able to detect different genetic patterns. In fact, within and among population diversity values observed vary across linguistic groups, with German and Italian speakers at the two extremes, and seem to reflect their different demographic histories. Using simulations we inferred that the joint effect of continued genetic isolation and reduced founding group size may explain the apportionment of genetic diversity observed in all groups. Extending the analysis to other continental populations, we observed that the genetic differentiation of Ladins and German speakers from Europeans is comparable or even greater to that observed for well known outliers like Sardinian and Basques. Finally, we found that in south Tyroleans, the social practice of Geschlossener Hof, a hereditary norm which might have favored male dispersal, coincides with a significant intra-group diversity for mtDNA but not for Y chromosome, a genetic pattern which is opposite to those expected among patrilocal populations. Together with previous evidence regarding the possible effects of "local ethnicity" on the genetic structure of German speakers that have settled in the eastern Italian Alps, this finding suggests that taking socio-cultural factors into account together with geographical variables and linguistic diversity may help unveil some yet to be understood aspects of the genetic structure of European populations.

New insights into the Tyrolean Iceman's origin and phenotype as inferred by whole-genome sequencing.

The Tyrolean Iceman, a 5,300-year-old Copper age individual, was discovered in 1991 on the Tisenjoch Pass in the Italian part of the Ötztal Alps. Here we report the complete genome sequence of the Iceman and show 100% concordance between the previously reported mitochondrial genome sequence and the consensus sequence generated from our genomic data. We present indications for recent common ancestry between the Iceman and present-day inhabitants of the Tyrrhenian Sea, that the Iceman probably had brown eyes, belonged to blood group O and was lactose intolerant. His genetic predisposition shows an increased risk for coronary heart disease and may have contributed to the development of previously reported vascular calcifications. Sequences corresponding to ~60% of the genome of Borrelia burgdorferi are indicative of the earliest human case of infection with the pathogen for Lyme borreliosis.

RNA-based analysis of BRCA1 and BRCA2 gene alterations.

Alterations in BRCA1 and BRCA2 genes account for a large proportion of hereditary breast and ovarian cancers. Mutations and variants of unknown pathological significance have been identified in both genes; however, most of them have been studied only at the genomic level, and their effect on mRNA expression remains unknown. We identified two BRCA1 and six BRCA2 splice site variants, and one BRCA2 alteration at exon 14. Our aim was to ascertain the effect on RNA processing of the variants still unclassified. We found that BRCA1 c.IVS11 + 1G>A, BRCA2 c.7252_7272delinsTG, BRCA2 c.IVS2 + 1G>A, BRCA2 c.IVS13-2A>G, BRCA2 c.IVS21 + 4A>G, and BRCA2 c.9345G>A lead to aberrant transcripts in lymphocytes. Five of these six splice site variants caused a complete inactivation of the mutant allele because they produced frameshift similar to previously described deleterious exonic variants. Therefore, we consider them to be true deleterious mutations, possibly associated with an increased lifetime risk of breast or ovarian cancer. BRCA1 c.IVS17 + 6C>G, BRCA2 c.IVS12-9del4, and BRCA2 IVS1-9del3 represent rare variants, not disrupting normal mRNA processing. The last two BRCA2 genetic variants had not been reported in the Breast Cancer Information Core BIC database.

Papillary lesions of the breast: a molecular progression?

INTRODUCTION: Breast papillary lesions represent a heterogeneous group of tumors ranging from benign to malignant, including several intermediate forms. Malignant papillary tumors are rare and their molecular characterization is still limited. A few studies pointed to the presence of specific genetic alterations that could be relevant both for diagnostic purposes and to elucidate tumour development and progression. In order to look into the issue, we compared LOH relative frequencies of four microsatellite markers located on chromosome 16 in a set of morphologically different papillary breast lesions. LOH at TP53 locus was also analyzed throughout lesions. MATERIALS AND METHODS: Fifteen cases were analyzed. Sections including a malignant papillary lesion, a benign lesion (when available), and normal breast tissue were selected. Fifteen malignant and twelve benign areas were microdissected using the Leica laser microdissection system (AS LMD). After DNA extraction samples were tested for the following markers: TP53, D16S423, D16S310, DS163210 and D16S476, and analyzed on ABI PRISM 3100 (Applied Biosystems, Foster city CA). RESULTS: Fourteen malignant lesions and twelve paired benign areas appeared to be informative for at least one of the four markers on chromosome 16. In particular, LOH at loci 16p13 and 16q21 was detected in both benign and malignant lesions, whereas LOH at locus 16q23 was limited to malignant lesions. Nine malignant and seven benign lesions were informative for LOH at TP53 locus, that was found to be significantly associated (p=0.01) with the malignant phenotype. CONCLUSIONS: Our data suggest an involvement of chromosome 16 mutations in the early steps of breast papillary tumorigenesis. TP53 deletion and possibly LOH at 16q23 appear to play a role as progression factors, being they significantly associated with malignant transformation of breast papilloma.

Clinicopathological significance of GADD45 gene alterations in human familial breast carcinoma.

GADD45 is a DNA damage responsive gene, induced following BRCA1 expression. Mutations at GADD45 might substitute for p53 alterations in hereditary breast tumours characterized by wild-type p53. We analyzed GADD45 alterations in 59 (15 BRCA-associated) familial breast carcinomas. No mutations were found. LOH at GADD45 locus was identified in 19/59 tumours. GADD45 does not appear to be a frequent mutational target in hereditary breast cancer.

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.

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.

p53 inactivation is a rare event in familial breast tumors negative for BRCA1 and BRCA2 mutations.

Germline mutations at BRCA1 or BRCA2 genes result in susceptibility to breast and ovarian cancers. BRCA1- and BRCA2-associated tumors have distinct histologic and molecular phenotypes, as compared to sporadic breast tumors. Typically, a higher grade of malignancy is observed in BRCA-associated cancers. A number of studies have suggested that BRCA1 and BRCA2 proteins are of importance in DNA repair and maintenance of genome integrity, bringing about molecular models of tumor pathogenesis. In particular, alterations at p53 gene have been suggested to be a necessary step in the tumorigenesis of BRCA-associated carcinomas. In fact, BRCA-associated breast cancers have higher p53 mutation frequencies than sporadic ones. At present, very little is known regarding BRCA non-associated familial tumors (termed BRCAx tumors). To our knowledge no data is available on p53 alterations in this sub-group of familial tumors. In this study p53 alteration frequencies were evaluated in 13 BRCA1, 11 BRCA2 and 55 BRCAx breast tumors. Tumor samples were analyzed for p53 gene mutations by PCR-SSCP/direct sequencing, and for p53 protein overexpression by immunohistochemistry (IHC). Altogether, p53 alterations were detected in 54% of BRCAI tumors compared with 5% of BRCAx tumors. No p53 alteration was found in BRCA2 tumors. While loss of p53 checkpoint control is likely to be an important step in the molecular pathogenesis of BRCA1-associated cancers, our data seem to indicate a p53-independent molecular mechanism underlying BRCAx neoplastic transformation.

Different expressivity of BRCA1 and BRCA2: analysis of 179 Italian pedigrees with identified mutation.

Mutations in BRCA1 and BRCA2 show different expressivity with respect to cancer risk, and allelic heterogeneity may be present in both genes. We collected 179 pedigrees with identified germline mutation (104 BRCA1 and 75 BRCA2), ascertained in six collaborating centers of the Italian Consortium for Hereditary Breast and Ovarian Cancer. Significant heterogeneity was detected for several variables, and a logistic regression model including age of diagnosis in the proband, presence of ovarian cancer in the family, presence of prostate or pancreatic cancer in the family, and presence of male breast cancer in the family proved to be effective in predicting the presence of a mutation in a gene rather than the other. Excess of familial aggregation of both breast and ovarian cancer was observed in both genes. Proportion of ovarian cancer was increased in the 5' portion of BRCA1, and presence of prostate or pancreatic cancer in a family was correlated with presence of ovarian cancer in BRCA2.