SCIENTIFIC REPORT 2012-‐2014 UMF „CAROL DAVILA

 SCIENTIFIC REPORT 2012-­‐2014 UMF „CAROL DAVILA” RESEARCH TEAM Dr. Florina Raicu Dr. Relu Cocos Dr. Sorina Schipor Dr. Laurentiu Camil Bohiltea REF. Revealing Bucharest’s past: an integrative study of ancient DNA and osteoarchaeological data of late medieval populations, Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PNII-­‐ID-­‐PCCE-­‐2011-­‐2-­‐0013 The genetic structure of a population is a mosaic of past and recent demographic changes. Population bottlenecks increase the rate of random genetic drift and increase inbreeding leading to the loss of genetic variation which may lead to lower levels of individual fitness, reduced resistance to parasites and diseases, and reduced ability to respond to environmental changes. Application of molecular genetics methods in human osteoarchaeology could retrieve new types of information and help the paleodemographical interpretation, past migrations pathways and population history. Our aim is to study the nature and extent of temporal changes in population genetic variation of the medieval Bucharest compared with nowadays Romanians based on the comparisons of the haplogroups or haplotypes frequencies of the mtDNA and the Y chromosome data. The medieval and modern genetic structure of the Romanian population could reveal strikingly different frequencies induced by short-­‐term historical European and Balkan processes (population replacement, admixture and minor migrations events due to lethal epidemics, wars, religious and cultural incidents) that are partly described in historical chronicles, archeological and anthropological sources. The archaeogenetics analysis relies on the study of ancient mitochondrial DNA and Y-­‐chromosome genetic markers. The distribution of the mtDNA types found in the ancient sample, by analyzing both coding and control regions of mitochondrial DNA, will unravel the changes of frequency haplotypes and the Y chromosome genetic diversity will be analyzed using a combination of slow and fast-­‐mutating polymorphisms, Y SNP and Y STR markers of the aDNA, in order to obtain a detailed view of Y markers variation in Bucharest paternal gene pool over four centuries (16th-­‐19th centuries AD). The analysis of rapidly evolving Y-­‐chromosomal short tandem repeat loci (Y-­‐STRs) reveals a signature of more recent historic events, not previously detected by other genetic markers. When high-­‐
resolution binary lineages are coupled to more rapidly mutating microsatellites than, the combination of linked polymorphic markers becomes a powerful tool for understanding diversity across different time frames and reconstruct the more recent genetic history of our studied populations. PROJECT WORKING PLAN FOR 2012-­‐2013 At the end of 16th month we will complete extraction of aDNA from 20 skeletons and perform quantitative PCR on the aDNA samples. Also, the HVR I mtDNA typing will be realized for 20 aDNA samples and 600 modern DNA using sequence analysis for control region PROJECT WORKING PLAN FOR 2014 At the end of 30th month we will complete extraction from the second lot of 50 bones samples and conduct quantitative PCR on the aDNA samples. Also, the mtDNA typing will be completed for other 50 aDNA samples and HVRII for 600 modern DNA. Starting the Y SNP typing for aDNA samples and the cloning mtDNA procedure. Preliminary data will be presented at international specific congresses and conferences. Page 1 of 6 PROJECT ACTIVTY REPORT FOR 2012-­‐2013 Ancient DNA extraction and authenticity criteria The ancient DNA extraction from bones is the most sensitive part of genetic protocol, thus genetics team members were trained in an international reference laboratory from University of the Basque Country. Working with ancient DNA is difficult and the crucial requirement to achieve reliable results is to provide a contamination free environment for handling and analyzing the historical samples. Ancient DNA undergoes to different kind of chemical damage during years, resulting in the shortening of DNA fragment and in the deamination (of C to U). Another important feature is the contamination from environmental, exogenous DNA and modern DNA; it was demonstrated that about 98% of DNA isolated from an ancient remain correspond to bacterial and fungal DNA. Moreover, if the ancient sample has human origins, the contamination from modern human DNA could dramatically affect the authenticity of the experiment results. Taken together, these characteristics of aDNA, could reduce the chance to obtain valuable results, so that researchers have outlined a series of guidelines to ensure the quality of ancient DNA data, resulting in the nine “gold criteria” outlined by Cooper and Poinar in “Ancient DNA: do it right or not at all”. In the absence of full compliance with all nine criteria, the reliability and authenticity of results remain uncertain. We built and outfitted in the first 4 month of the project an Ancient DNA Laboratory with spatially and temporally separated rooms for aDNA extraction and PCR amplification to specially facilitate extraction and analyzing of ancient DNA. The aDNA lab was built in the Romanian Institute of Anthropology from Bucharest. We process all ancient samples originating from human sources in our ‘clean room’. We clean all the surfaces and equipments with UV light and sodium hypochlorite, using of disposable gloves, lab coats, caps, shoe covers and masks as standard procedures. Samples are prepared and extracted individually. Samples are cleaned (with successive washes of 10% sodium hypochlorite, ethanol, and sterile water) and UV irradiated. Before sampling, in order to eliminate surface contamination, a 1-­‐2-­‐mm layer of the outer surface of the bones is removed by transversal hemi-­‐sections with Dremel MultiPro tool and collected in a tube. For tooth samples, the tooth is cut in half exposing both the crown and the root. Some of the bone samples are pulverized under liquid nitrogen in a freezer mill SPEX Sample Prep 6751 and other are extracted from teeth using an internal new method without grinding especially developed for speeding up the work. When grinded is used up to 100 mg bone powder were demineralized overnight using 0.5 M EDTA and lized with proteinase K. Samples before processing Samples after DNA extraction All pre-­‐PCR manipulation and cutting of teeth, extraction of DNA and mixing of reactions for PCR are also performed in our ‘‘Clean-­‐Laboratory’’ dedicated solely to aDNA work. DNA extraction is performed in laminar flow box. Multiple reagent blanks and negative control reactions are performed with all amplifications. At least 3 independent DNA extracts are performed. Ancient DNA was isolated from 70 skeletal remains (teeth with closed apex still sitting firmly in the alveolus) of individuals excavated at Piata Universitatii archaeological site in Bucharest dated to 16th century. Page 2 of 6 Contemporary DNA was extracted from bloods with PureLink Genomic DNA kit according to the protocol of the manufacturer from 700 biological samples. aDNA and modern DNA extraction was a continuous proces during the first and the second year of the project. After aDNA extraction all the samples are concentrated and purified. In order to destroy unwanted DNA fragments before each experiment all steps involving DNA extraction and PCR mixes occur in the clean room, which is equipped with a special HEPA filter and UV lamps. This room is under positive airflow to keep exogenous DNA from entering the room. For the PCR process, several working dilutions with varying amounts of BSA were tried and negative controls were included in all PCRs. A minimum of 3 amplifications is carried out from different extracts of the same sample to increase the confidence in the results. Our PCR and post-­‐PCR laboratories are placed in two different locations to avoid any contamination. Our post-­‐PCR lab is located in the 'Carol Davila' University of Medicine and Pharmacy, Chair of Medical Genetics. This lab houses all the equipment needed to amplify ancient DNA, quantify, clone and sequence aDNA. The lab incorporates also instrumentation, lab space and reagents, which encompasses work carried out by many researchers, allowing us to perform different studies on contemporary DNA such as analyzing various genetic diseases. DNA quantification We used our standard procedure to quantify aDNA, which consists on measuring the number of molecules of a segment of 113 bp (including primers; 73 bp without primers) of HVS-­‐I of mtDNA by means of qPCR (Step-­‐One, Life Technologies). For this, we used a set of primers and a TaqMan probe (TaqMan Gene Expression Master Mix, Life Technologies) si SYBR Green (SYBR Green PCR Master MIX, Invitrogen). We obtained between 500 and 140000 copies. All consumables are purchased as DNA-­‐clean and are further UV irradiated before use for at least 10 minutes in a UV crosslinker. For nuclear DNA quantitative PCR (qPCR) is conducted on the aDNA bone extracts using PCR kit Quantifiler® Human DNA Quantification Kit (Life Technologies) or SYBR Green PCR master-­‐mix (Life Technologies) on Real Time PCR. PROJECT ACTIVTY REPORT FOR 2014 Sequencing Sequences from the two hypervariable regions (HVS-­‐I and HVS-­‐II) of the control region of the mitochondrial DNA spanning positions 16024-­‐16569 and 1-­‐576 were obtained until now by analyzing 70 aDNA samples out of a total of 150 dental pieces recovered from the skeletons excavated from the cemetery and 700 modern DNA samples collected. Hypervariable regions I and II of ancient mtDNA were amplified and sequenced using twelve overlapping fragments, each with a length of approximately 100 base pairs. Amplicons were purified with Qiaquick columns and directly sequenced under manufacturer’s cycle sequencing parameters by cycle sequencing with BigDye Terminator v1.1 Cycle Sequencing Kit for aDNA and BigDye Terminator v3.1 Cycle Sequencing Kit for contemporary DNA using the same primers as for the amplification. Sequencing products were purified DyeEx 2.0 Spin kit following the manufacturer’s recommendations. Capillary electrophoresis will be carried out on the 310 Genetic Analyzer and sequencing reaction results were analyzed with the SeqScape softwareTM v2.5 . Sequence evaluation was performed by two independent scientists. Analysis of mitochondrial DNA sequencing data was performed using the ABI PRISM DNA Sequencing Analysis Software Version 3.7 and sequences were aligned and compared with the revised Cambridge Reference Sequence rCRS using the SeqScape Software Version 2.5 and MEGA5. Haplogroups were assigned using HaploGrep software. Page 3 of 6 mtDNA profiles analysis Since the Middle Ages, Romanian population lived in three distinct provinces Wallachia, Moldavia and Transylvania until the 19th century. Over the centuries, their territories were repeatedly invaded by different peoples and subjected to external political influences resulting in demographic changes that could affected the genetic structure of populations. Very little work has been done on getting specific information of mtDNA in Romanian population. We performed a large-­‐scale mitochondrial DNA analysis from our historical provinces in order to visualize the relationships between Romanian and other populations Europe based on 700 HVSI and HVS II mtDNA sequence data. The mitochondrial DNA (mtDNA) sequences of Europeans are sorted into nine major phylogenetic clades, or haplogroups, alphabetically named H, J, K, N1, T, U4, U5, V, X, and W which all belong to macrohaplogroup N part of L3 clade. Haplogroup H alone constitutes about one half of the European mtDNA pool. The analyses of population relationships based on mtDNA showed that most of the Balkan populations form a homogeneous set and are similar to surrounding European populations. Haplogroup U is held as the oldest maternal haplogroup found and was the dominant type of mtDNA in Europe before the spread of agriculture into region. Haplogroup W is particularly common in the eastern half of Europe, in the North Caucasus, in Central Asia, in Iran and in the north-­‐west of India. The highest frequencies worldwide of macrohaplogroup M are observed in Asia where frequencies range from 60%-­‐
80%. The majority of the ancient and modern mtDNA samples analyzed by now fall into the common West Eurasian mitochondrial haplogroups. All nine common European haplogroups (H, I, J, K, T, U, V, W, and X) were observed in our samples, and these were divided into subgroups when possible. The most common haplogroup in Bucharest region was H as it is common in Europe. The other common haplogroups were U, T, HV, J, N and W. The haplogroups observed less frequently include K, I, R, X, M and D and these were observed in approximately 2% or less of the samples. The results showed that present day Romanians in all provinces share their maternal ancestry with both eastern/central European and Balkan populations. The three populations of Romania analyzed here exhibit slightly different mtDNA lineage compositions, mainly consisting of the haplogroups H, U, J, T, K, N and W, with significant frequency differences corresponding for H, U, M and W haplogroups. Overall, this study provides a first comprehensive analysis of mtDNA genome variation in Romania revealing the existence of different degrees of provincial differences of haplogroup frequencies. A special feature of our samples was a higher frequency of the haplogroups W and M, which are relatively small groups in Europe. We analyzed mtDNA variation using 50 aDNA samples from Piata Universitatii archaeological site and 700 modern DNA to study nature and extent of temporal changes in genetic variation in Bucharest region during 16th-­‐19th centuries. The archaeological site of Piata Universitatii cemetery unearthed about 676 graves with 900 skeletons.The reduced number of aDNA samples analyzed by now could not offer yet enough support for statistical analysis. The majority of the ancient and modern mtDNA samples analyzed by now falls into the common West Eurasian mitochondrial haplogroups. In conclusion, to fully assess the dynamics of the historical population composition by comparing genotypes in a temporal context we have to complete the comparative analysis of all aDNA and more modern DNA samples. Moreover, in order to reveal possible genetic data changes caused by a possible population bottleneck corresponding to the waves of lethal epidemics, in which an almost one-­‐third of the population was lost, in 2015 we will also investigate a set of 17 fast evolving short tandem repeat loci (STR) and we will study also some isolated populations from our country. Page 4 of 6 Statistics Pairwise populations genetic distances were calculated in Arlequin ver3.5 for mtDNA data. Tajima’s D statistics and Fu and Li’s D and F statistics were calculated using the DnaSP program. In order to characterize how populations cluster based on genetic variation we performed Multidimensional scaling (MDS) using the pairwise Fst values. To determine the relationships between the surrounding and other European populations we also performed PCA analysis using SPSS 20 software. Contour maps representing geographic distributions of W, U and M haplogroups frequencies across Europe were created using the Kriging method in Surfer 9 Golden software. Areas of high frequencies are represented by darker tone of the blue colour used in the maps. FREQUENCY ANALYSIS OF THE CCR5-­‐DELTA32 ALLELE IN MEDIEVAL AND MODERN ROMANIAN POPULATION The CCR5-­‐delta 32 mutation in human chemokine receptor gene can be considered a rare example of a beneficial mutation. The mutation gives its homozygous carriers complete resistance against HIV infection and has been proposed to provide protection against different lethal epidemics. Although the origin of the mutation indicates prehistoric times, repeated waves of lethal epidemics over medieval centuries strongly modified the European gene pool via bottleneck effect and seem to increase the frequency of CCR5-­‐delta 32 up to 20% as a result of selective advantage. High allele frequencies are found in Scandinavian countries, and the lowest rates in Europe are found in Sicily and the Iberian Peninsula There is a general agreement that genetic drift was not responsible and a heterozygote advantage led to the spreading of the mutant allele in Europe. In order to investigate if the lethal epidemics of the medieval times selected the allele we analyzed the presence of this mutation in aDNA isolated from the skeletons discovered in Piata Universitatii archeological site and compared them to the frequency in a control group from local modern population. CCR5 PCR primers were designed to amplify a 130 bp fragment and 98-­‐bp for Δccr5. Our preliminary results based on 50 aDNA samples and 700 modern DNA indicate that the frequency of the allele in medieval Romanian population is not statistically significant when compared to contemporary one suggesting that lethal epidemics had little effect on its present day frequency. The oberved frequency of medieval samples is not statistically significant due to reduced numbers of analyzed samples until now. We can not conclude based on preliminary data that increase in the allelic frequency of the mutant CCR5-­‐delta 32 allele has been positive selected before the medieval centuries. We typed 50 individuals from archaeological site and found the frequency of CCR5/CCR5 -­‐D32 to be 5,2 %. We also typed the individuals from contemporary control group and obtained a frequency of CCR5/CCR5 D32 to be 9,22% and 0.7% of CCR5 D32/CCR5 D32 for all Romanian provinces, 6.35% for Bucharest region. Based on preliminary results we identify a significative diference between Bucharest and the rest of Romanian populations sugesting the idea of a population with specific continuous genetic structure from medeval times. Page 5 of 6 PUBLISHED PAPERS IN THE FIELD: GENETIC STRUCTURE OF MEDIEVAL AND MODERN ROMANIAN POPULATION Articles 1. Cocoş R, Şendroiu A, Schipor S, Bohîlţea LC, Şendroiu I, et al. (2014) Genotype-­‐Phenotype Correlations in a Mountain Population Community with High Prevalence of Wilson’s Disease: Genetic and Clinical Homogeneity. PLoS ONE 9(6): e98520. doi 10.1371/journal.pone.0098520 2. Varzari A, Kharkov V, Nikitin AG, Raicu F, Simonova K, Stephan W, Weiss EH, Stepanov V, PLoS One., Paleo-­‐balkan and slavic contributions to the genetic pool of moldavians: insights from the y chromosome, 2013;8(1):e53731. doi: 10.1371/journal.pone.0053731. Presentations at International Conferences: 3. F. Raicu, R. Cocos, S. Schipor, L. Bohiltea, „Frequency analysis of the CCR5-­‐Delta32 allele in medieval and modern Romanian population”, EUROPEAN JOURNAL OF HUMAN GENETICS, Vol. 21 Supp. 2, June 2013, p. 381. 4. R. Cocos, S. Schipor, L. Bohiltea, F. Raicu, „Mitochondrial DNA diversity in medieval and modern Romanian Population”, EUROPEAN JOURNAL OF HUMAN GENETICS, Vol. 21 Supp. 2, June 2013, p. 381. 5. Relu Cocos, Laurentiu Bohiltea, Sorina Schipor, Florina Raicu EUROPEAN JOURNAL OF HUMAN GENETICS “Mitochondrial DNA variation analysis in historical provinces of Romania”, Vol. 22 Supp. 1, May 2014, p. 327. Thesis 1. Postdoc thesis, dr. Sorina Schipor, research team member, Postdoc Program in Biomedicine and Biotechnology POSDRU/81/3.2/S/55362, University of Bucuresti, Faculty of Biology, Title in Romanian: „ANALIZA GENEI RECEPTORULUI CCR5 (CCR5-­‐DELTA32) LA POPULAŢIA DIN ROMÂNIA” 2. Graduated student UMF Carol Davila, Munteanu C. Ion, Title in Romanian ”STUDIU DE ASOCIERE PRIVIND HAPLOTIPURILE ADN-­‐ULUI MITOCONDRIAL SI FRECVENTA MUTATIEI CCRDELTA 32” Page 6 of 6