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Population genetics of Turkish Cypriots from Cyprus: Forensic and anthropological implications
Accepted Manuscript Title: Population genetics of Turkish Cypriots from Cyprus: forensic and anthropological implications Author: C. Gurkan D.K. Demirdov H. Sevay PII: DOI: Reference:
S1875-1768(15)30028-7 http://dx.doi.org/doi:10.1016/j.fsigss.2015.09.152 FSIGSS 1149
To appear in: Received date: Accepted date:
11-8-2015 7-9-2015
Please cite this article as: C.Gurkan, D.K.Demirdov, H.Sevay, Population genetics of Turkish Cypriots from Cyprus: forensic and anthropological implications, Forensic Science International: Genetics Supplement Series http://dx.doi.org/10.1016/j.fsigss.2015.09.152 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Population genetics of Turkish Cypriots from Cyprus: forensic and anthropological implications C. Gurkana* [email protected], D. K. Demirdova, H. Sevayb a
Turkish Cypriot DNA Laboratory, Nicosia, North Cyprus
b
Department of Information Systems Engineering, Near East University, Nicosia, North Cyprus
*Corresponding author. Tel.: +905428584783; fax: +903922238856
1
ABSTRACT Turkish Cypriot DNA Laboratory (TCDL) operates under the Turkish Cypriot Member Office of the Committee on Missing Persons in Cyprus (CMP). TCDL contributes to the UN-led CMP Project on Exhumation, Identification and Return of Remains of Missing Persons and also plays a leading role in the establishment of forensic genetic services in North Cyprus. A family reference sample (FRS) bank comprising the relatives of the Turkish Cypriot missing persons (MiPs) from the 1963/64 and 1974 era was established first. Since 2012, TCDL assumed full responsibility for DNA typing of all Turkish Cypriot FRSs, which are then used for familial DNA matching with those for the skeletal remains exhumed from all over Cyprus and analyzed at international laboratories such as ICMP and Bode Technology. Using a second sample bank comprising Turkish Cypriot volunteer samples, TCDL also conducts population studies for requisite statistical evaluations of MiP identifications.
Keywords: missing persons; variant alleles; multi-allelic patterns; FRS.
2
1. Introduction Cyprus is the third largest island in the Mediterranean Sea uniquely situated at the crossroads of civilizations throughout history. As a result, Cyprus has a multi-ethnic population structure strictly defined by different native languages and religious denominations. Constituting the second most populous ethnic group on the island, Turkish Cypriots largely adhere to the Sunni sect of Islam and speak Turkish. Tracing their origins mainly to the Ottoman Conquest of the island in 1571, Turkish Cypriots used to reside all over the island in ethnically mixed or exclusive settlements up until 1963/64 and 1974 when the inter-communal strife intensified. Following the de facto division of the island in 1974, Turkish Cypriots today largely reside in North Cyprus. According to the Committee on Missing Persons (CMP) in Cyprus (http://www.cmpcyprus.org/), there are 493 Turkish Cypriot and 1508 Greek Cypriot missing persons (MiPs) as a result of the tragic events of 1963/64 and 1974. CMP operates an ambitious project called ‘Exhumation, Identification and Return of Remains of Missing Persons’ where MiP identifications are now made through scientific reconciliation of archaeological, anthropological and genetic evidence. Operating directly under the CMP Turkish Cypriot Member Office, the Turkish Cypriot DNA Laboratory (TCDL) contributes to the CMP project through the collection and subsequent DNA profiling of (a) the family reference samples (FRSs) corresponding to the Turkish Cypriot MiPs for identification purposes and (b) Turkish Cypriot volunteer samples so that population genetics studies could be conducted for requisite statistical assessment of MiP identifications.
2. Materials and methods Blood/buccal swab samples were collected with informed consent, and subsequent genomic DNA extractions were carried out using the Life Technologies PureLinkTM Genomic DNA Mini Kit. While FRSs were collected mainly from close genetic relatives of MiPs, spouses were also sampled when a given MiP and his/her spouse had offspring(s). For population studies, volunteers, who were not MiP relatives, were selected from among those that were not only born in Cyprus and/or were residing in North Cyprus, but also with both parents born in Cyprus. 15 autosomal short tandem repeat (STR) loci and/or 17 Y-chromosomal STR (Y-STR) loci were PCR amplified using the Life Technologies AmpFLSTR® IdentifilerTM and YfilerTM Systems. To allow full compatibility with other popular autosomal STR systems (e.g., Promega PowerPlex® 16), an in-house tri-plex system comprising three autosomal mini STR loci [D21S11, Penta E and Penta D] was also used [1]. Capillary gel electrophoresis was performed using the ABI 3130 Genetic Analyzer. All DNA extractions and STR haplotyping were conducted at TCDL, whose proficiency has been certified through the YHRD Y-STR Haplotyping Quality Assurance Exercise 2013 (17 Y-STR loci)
3
and the International Society for Forensic Genetics (ISFG) English Speaking Working Group Relationship Testing Workshop 2015 (17 autosomal STR loci and 17 Y-STR loci).
3. Results and discussion Table 1 summarizes the number of Turkish Cypriot FRSs and other volunteer samples analyzed at TCDL to date. To emphasize the genetic diversity of the Turkish Cypriot samples in general, the number and different types of allelic variants and multi-allelic patterns, as well as potential primer binding site mutation (PBSM) and mosaicism cases observed are also tabulated. Figure 1 illustrates the precise geographical distribution of the paternal and maternal birthplaces for the 501 Turkish Cypriot volunteers included in the 15-loci autosomal STR population study [2]. A similar geographic distribution was observed for the paternal origins of 253 Turkish Cypriot volunteers included in the 17-loci Y-STR population study [3]. An expanded version of the Turkish Cypriot 17-loci Y-STR dataset (n=380) along with in silico haplogroup assignments has also become available (Gurkan et al., submitted). Furthermore, a subset (n=60) of the Turkish Cypriot volunteer samples was analyzed elsewhere using 55 ancestry informative single nucleotide polymorphisms (AISNP) markers [4]. Cumulative results from the autosomal and Y-chromosomal STR based population studies suggest that while the Turkish Cypriots may have a close genetic connection with the Levant and Southeastern European populations at least through their paternal lineages, they have an even stronger genetic connection with the Anatolian populations based on both autosomal and Y-STR data available [2, 3]. At the same time, Turkish Cypriots were also found to possess very rare, if not novel, genetic characteristics such as allelic variants and multi-allelic patterns that were seemingly exclusive to this population alone.
Acknowledgements We thank Mrs. Gulden Plumer Kucuk, the Turkish Cypriot Member of CMP, and her office for financial and administrative support and Dr. Carlos Vullo at LIDMO-EAAF for kindly providing the protocol for the in-house tri-plex system. C. Gurkan’s attendance at ISFG2015 was partially sponsored by the North Cyprus Forensic Sciences Association (Kuzey Kıbrıs Adli Bilimler Derneği KABiDer).
Conflict of interest None.
4
References [1] J.M. Butler, Y. Shen, B.R. McCord, The development of reduced size STR amplicons as tools for analysis of degraded DNA, J Forensic Sci 48 (2003) 1054-1064. [2] C. Gurkan, D.K. Demirdov, R.F. Yamaci, et al., Population genetic data for 15 autosomal STR markers in Turkish Cypriots from Cyprus, Forensic Sci Int Genet 14 (2015) e1-e3. [3] K. Terali, T. Zorlu, O. Bulbul, et al., Population genetics of 17 Y-STR markers in Turkish Cypriots from Cyprus, Forensic Sci Int Genet 10 (2014) e1-e3. [4] A.J. Pakstis, E. Haigh, L. Cherni, et al., 52 Additional Reference Population Samples for the 55 AISNP panel, Forensic Sci Int Genet (2015) (in press).
5
Figure Captions
Figure 1. Distribution of the volunteer samples by paternal and maternal birthplaces for the Turkish Cypriot autosomal STR population study (n=501) from Cyprus [2]. Blue, red and green circles denote settlements associated with paternal birthplaces alone, maternal birthplaces alone, and both paternal and maternal birthplaces (at least one each, albeit not necessarily for the same volunteer) in that order. The size of each circle relatively indicates the total number of paternal and/or maternal birthplaces associated with a given geographic location. Self-stated paternal and maternal birthplaces for each volunteer were queried through the Google Maps application programming interface (https://www.google.com/maps) to obtain the corresponding precise geographic coordinates, which were then plotted on the map using an in-house web application.
6
Tables Table 1. Turkish Cypriot FRSs and other volunteer samples analysed at TCDL to date and various genetic variants observed Sample Type: Analysis Aim: Systems Used Number of Samples Analysed (n)
Allelic Variants Observed
Multi-Allelic Patterns Observed Potential Mosaicism Case
Turkish Cypriot Family Reference Samples (FRSs) Missing Person Identifications
Turkish Cypriot Volunteer Samples Population Genetics Studies
Identifiler
in house Penta E/Penta D/D21S11
Yfiler
Identifiler
in house Penta E/Penta D/D21S11
Yfiler
(15 autosomal loci)
(3 autosomal loci)
(17 Y-STR loci)
(15 autosomal loci)
(3 autosomal loci)
(17 Y-STR loci)
406
375
26
501 a
501 b
253 c / 380 d
-
1x Penta D*6 2x Penta E*12.2 5x Penta E*16.4 3x Penta E*17.4 -
1x DYS458*18.2 1x DYS458*19.2 1x DYS458*20.2 -
-
-
1x amelogenin-X:Y unusual h peak height imbalance b
c
1x D21S11*31.3 2x D21S11*34.1 e 1x D21S11*25.3 1x D21S11*25.3 PBSM 1x D19S433*13.1 2x Penta D*6 2x D2S1338*13 2x Penta E*14.4 1x FGA*25.1 1x Penta E*15.4 5x Penta E*17.4 2x Penta E*18.4 -
-
f
1x DYS385a/b*12.2,18 f 1x DYS385a/b*12,13.2 3x DYS392*10.2 1x DYS437*12 f,g 3x DYS438*9.4 3x DYS456*12 1x DYS458*12 10x DYS458*13 g 1x DYS458*14.3 1x DYS458*16.2 9x DYS458*17.2 15x DYS458*18.2 11x DYS458*19.2 3x DYS458*20.2 2x DYS458*21 2x DYS635*17 1x DYS635*27 1x Y-GATA-H4*14 2x DYS19*15,16 f 1x DYS438*9,10 f 1x DYS385a/b*11,13,17 f 1x DYS385a/b*12,13,17 -
a
Gurkan et al. (2015) FSI Genetics 14, e1-e3; Gurkan et al. (unpublished); Terali et al. (2014) FSI Genetics 10, e1-e3 (n=253) and Gurkan et al. (submitted) (n=380)
e
Primer binding site mutation (PBSM) @ D21S11*25.3 was detected by comparing results from the Identifiler and PentaE/PentaD/D21S11 systems for the same sample.
d
f
Allelic variants, multi-allelic patterns and genotypes that were not yet reported elsewhere according to YHRD, STRBase and the current literature
g
These allelic variants were confirmed by double-stranded DNA sequencing by Becky Hill and Peter Valone at NIST
h
Identifiler amelogenin X and Y signal ratio of 5:1 was observed despite normal Yfiler profile for the same sample, see Dauber et al. (2004) Int Congr Ser 1261,508-510 for further discussion
7
S1875-1768(15)30028-7 http://dx.doi.org/doi:10.1016/j.fsigss.2015.09.152 FSIGSS 1149
To appear in: Received date: Accepted date:
11-8-2015 7-9-2015
Please cite this article as: C.Gurkan, D.K.Demirdov, H.Sevay, Population genetics of Turkish Cypriots from Cyprus: forensic and anthropological implications, Forensic Science International: Genetics Supplement Series http://dx.doi.org/10.1016/j.fsigss.2015.09.152 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Population genetics of Turkish Cypriots from Cyprus: forensic and anthropological implications C. Gurkana* [email protected], D. K. Demirdova, H. Sevayb a
Turkish Cypriot DNA Laboratory, Nicosia, North Cyprus
b
Department of Information Systems Engineering, Near East University, Nicosia, North Cyprus
*Corresponding author. Tel.: +905428584783; fax: +903922238856
1
ABSTRACT Turkish Cypriot DNA Laboratory (TCDL) operates under the Turkish Cypriot Member Office of the Committee on Missing Persons in Cyprus (CMP). TCDL contributes to the UN-led CMP Project on Exhumation, Identification and Return of Remains of Missing Persons and also plays a leading role in the establishment of forensic genetic services in North Cyprus. A family reference sample (FRS) bank comprising the relatives of the Turkish Cypriot missing persons (MiPs) from the 1963/64 and 1974 era was established first. Since 2012, TCDL assumed full responsibility for DNA typing of all Turkish Cypriot FRSs, which are then used for familial DNA matching with those for the skeletal remains exhumed from all over Cyprus and analyzed at international laboratories such as ICMP and Bode Technology. Using a second sample bank comprising Turkish Cypriot volunteer samples, TCDL also conducts population studies for requisite statistical evaluations of MiP identifications.
Keywords: missing persons; variant alleles; multi-allelic patterns; FRS.
2
1. Introduction Cyprus is the third largest island in the Mediterranean Sea uniquely situated at the crossroads of civilizations throughout history. As a result, Cyprus has a multi-ethnic population structure strictly defined by different native languages and religious denominations. Constituting the second most populous ethnic group on the island, Turkish Cypriots largely adhere to the Sunni sect of Islam and speak Turkish. Tracing their origins mainly to the Ottoman Conquest of the island in 1571, Turkish Cypriots used to reside all over the island in ethnically mixed or exclusive settlements up until 1963/64 and 1974 when the inter-communal strife intensified. Following the de facto division of the island in 1974, Turkish Cypriots today largely reside in North Cyprus. According to the Committee on Missing Persons (CMP) in Cyprus (http://www.cmpcyprus.org/), there are 493 Turkish Cypriot and 1508 Greek Cypriot missing persons (MiPs) as a result of the tragic events of 1963/64 and 1974. CMP operates an ambitious project called ‘Exhumation, Identification and Return of Remains of Missing Persons’ where MiP identifications are now made through scientific reconciliation of archaeological, anthropological and genetic evidence. Operating directly under the CMP Turkish Cypriot Member Office, the Turkish Cypriot DNA Laboratory (TCDL) contributes to the CMP project through the collection and subsequent DNA profiling of (a) the family reference samples (FRSs) corresponding to the Turkish Cypriot MiPs for identification purposes and (b) Turkish Cypriot volunteer samples so that population genetics studies could be conducted for requisite statistical assessment of MiP identifications.
2. Materials and methods Blood/buccal swab samples were collected with informed consent, and subsequent genomic DNA extractions were carried out using the Life Technologies PureLinkTM Genomic DNA Mini Kit. While FRSs were collected mainly from close genetic relatives of MiPs, spouses were also sampled when a given MiP and his/her spouse had offspring(s). For population studies, volunteers, who were not MiP relatives, were selected from among those that were not only born in Cyprus and/or were residing in North Cyprus, but also with both parents born in Cyprus. 15 autosomal short tandem repeat (STR) loci and/or 17 Y-chromosomal STR (Y-STR) loci were PCR amplified using the Life Technologies AmpFLSTR® IdentifilerTM and YfilerTM Systems. To allow full compatibility with other popular autosomal STR systems (e.g., Promega PowerPlex® 16), an in-house tri-plex system comprising three autosomal mini STR loci [D21S11, Penta E and Penta D] was also used [1]. Capillary gel electrophoresis was performed using the ABI 3130 Genetic Analyzer. All DNA extractions and STR haplotyping were conducted at TCDL, whose proficiency has been certified through the YHRD Y-STR Haplotyping Quality Assurance Exercise 2013 (17 Y-STR loci)
3
and the International Society for Forensic Genetics (ISFG) English Speaking Working Group Relationship Testing Workshop 2015 (17 autosomal STR loci and 17 Y-STR loci).
3. Results and discussion Table 1 summarizes the number of Turkish Cypriot FRSs and other volunteer samples analyzed at TCDL to date. To emphasize the genetic diversity of the Turkish Cypriot samples in general, the number and different types of allelic variants and multi-allelic patterns, as well as potential primer binding site mutation (PBSM) and mosaicism cases observed are also tabulated. Figure 1 illustrates the precise geographical distribution of the paternal and maternal birthplaces for the 501 Turkish Cypriot volunteers included in the 15-loci autosomal STR population study [2]. A similar geographic distribution was observed for the paternal origins of 253 Turkish Cypriot volunteers included in the 17-loci Y-STR population study [3]. An expanded version of the Turkish Cypriot 17-loci Y-STR dataset (n=380) along with in silico haplogroup assignments has also become available (Gurkan et al., submitted). Furthermore, a subset (n=60) of the Turkish Cypriot volunteer samples was analyzed elsewhere using 55 ancestry informative single nucleotide polymorphisms (AISNP) markers [4]. Cumulative results from the autosomal and Y-chromosomal STR based population studies suggest that while the Turkish Cypriots may have a close genetic connection with the Levant and Southeastern European populations at least through their paternal lineages, they have an even stronger genetic connection with the Anatolian populations based on both autosomal and Y-STR data available [2, 3]. At the same time, Turkish Cypriots were also found to possess very rare, if not novel, genetic characteristics such as allelic variants and multi-allelic patterns that were seemingly exclusive to this population alone.
Acknowledgements We thank Mrs. Gulden Plumer Kucuk, the Turkish Cypriot Member of CMP, and her office for financial and administrative support and Dr. Carlos Vullo at LIDMO-EAAF for kindly providing the protocol for the in-house tri-plex system. C. Gurkan’s attendance at ISFG2015 was partially sponsored by the North Cyprus Forensic Sciences Association (Kuzey Kıbrıs Adli Bilimler Derneği KABiDer).
Conflict of interest None.
4
References [1] J.M. Butler, Y. Shen, B.R. McCord, The development of reduced size STR amplicons as tools for analysis of degraded DNA, J Forensic Sci 48 (2003) 1054-1064. [2] C. Gurkan, D.K. Demirdov, R.F. Yamaci, et al., Population genetic data for 15 autosomal STR markers in Turkish Cypriots from Cyprus, Forensic Sci Int Genet 14 (2015) e1-e3. [3] K. Terali, T. Zorlu, O. Bulbul, et al., Population genetics of 17 Y-STR markers in Turkish Cypriots from Cyprus, Forensic Sci Int Genet 10 (2014) e1-e3. [4] A.J. Pakstis, E. Haigh, L. Cherni, et al., 52 Additional Reference Population Samples for the 55 AISNP panel, Forensic Sci Int Genet (2015) (in press).
5
Figure Captions
Figure 1. Distribution of the volunteer samples by paternal and maternal birthplaces for the Turkish Cypriot autosomal STR population study (n=501) from Cyprus [2]. Blue, red and green circles denote settlements associated with paternal birthplaces alone, maternal birthplaces alone, and both paternal and maternal birthplaces (at least one each, albeit not necessarily for the same volunteer) in that order. The size of each circle relatively indicates the total number of paternal and/or maternal birthplaces associated with a given geographic location. Self-stated paternal and maternal birthplaces for each volunteer were queried through the Google Maps application programming interface (https://www.google.com/maps) to obtain the corresponding precise geographic coordinates, which were then plotted on the map using an in-house web application.
6
Tables Table 1. Turkish Cypriot FRSs and other volunteer samples analysed at TCDL to date and various genetic variants observed Sample Type: Analysis Aim: Systems Used Number of Samples Analysed (n)
Allelic Variants Observed
Multi-Allelic Patterns Observed Potential Mosaicism Case
Turkish Cypriot Family Reference Samples (FRSs) Missing Person Identifications
Turkish Cypriot Volunteer Samples Population Genetics Studies
Identifiler
in house Penta E/Penta D/D21S11
Yfiler
Identifiler
in house Penta E/Penta D/D21S11
Yfiler
(15 autosomal loci)
(3 autosomal loci)
(17 Y-STR loci)
(15 autosomal loci)
(3 autosomal loci)
(17 Y-STR loci)
406
375
26
501 a
501 b
253 c / 380 d
-
1x Penta D*6 2x Penta E*12.2 5x Penta E*16.4 3x Penta E*17.4 -
1x DYS458*18.2 1x DYS458*19.2 1x DYS458*20.2 -
-
-
1x amelogenin-X:Y unusual h peak height imbalance b
c
1x D21S11*31.3 2x D21S11*34.1 e 1x D21S11*25.3 1x D21S11*25.3 PBSM 1x D19S433*13.1 2x Penta D*6 2x D2S1338*13 2x Penta E*14.4 1x FGA*25.1 1x Penta E*15.4 5x Penta E*17.4 2x Penta E*18.4 -
-
f
1x DYS385a/b*12.2,18 f 1x DYS385a/b*12,13.2 3x DYS392*10.2 1x DYS437*12 f,g 3x DYS438*9.4 3x DYS456*12 1x DYS458*12 10x DYS458*13 g 1x DYS458*14.3 1x DYS458*16.2 9x DYS458*17.2 15x DYS458*18.2 11x DYS458*19.2 3x DYS458*20.2 2x DYS458*21 2x DYS635*17 1x DYS635*27 1x Y-GATA-H4*14 2x DYS19*15,16 f 1x DYS438*9,10 f 1x DYS385a/b*11,13,17 f 1x DYS385a/b*12,13,17 -
a
Gurkan et al. (2015) FSI Genetics 14, e1-e3; Gurkan et al. (unpublished); Terali et al. (2014) FSI Genetics 10, e1-e3 (n=253) and Gurkan et al. (submitted) (n=380)
e
Primer binding site mutation (PBSM) @ D21S11*25.3 was detected by comparing results from the Identifiler and PentaE/PentaD/D21S11 systems for the same sample.
d
f
Allelic variants, multi-allelic patterns and genotypes that were not yet reported elsewhere according to YHRD, STRBase and the current literature
g
These allelic variants were confirmed by double-stranded DNA sequencing by Becky Hill and Peter Valone at NIST
h
Identifiler amelogenin X and Y signal ratio of 5:1 was observed despite normal Yfiler profile for the same sample, see Dauber et al. (2004) Int Congr Ser 1261,508-510 for further discussion
7