Materials and methods
Chapter 1: Autosomal markers
3. Genetic portrait of Jewish populations based on three sets of X-chromosome markers: Indels, Alus and STRs
Ferragut JF; Bentayebi K; Pereira R; Castro JA; Amorim A; Ramon C and Picornell A
Forensic Science International: Genetics (submitted)
89 Introduction
The second chapter is devoted to the X-chromosome, since it is increasingly important in Forensic and Populations Genetics. On the one hand, the results of the X-chromosome combined with those of Y-chromosome and mtDNA will enable us to assess the paternal and maternal contribution to the genetic pool of Chuetas from the original Sephardic population and from the people among whom they lived (the gentile Majorcan population).
On the other hand, as has been explained in the introduction, X-chromosome markers play an increasing role in Forensic Genetics, especially to solve complex kinship cases.
The first set of markers we studied was the 12 X-STRs of Investigator Argus X-12 Kit (Qiagen) due to its growing use in Forensic Genetics in the last years. Since it had not been studied before, first of all we characterized the host population of the Chuetas, Majorca and its neighbouring populations (Minorca, Ibiza and Valencia) (Ferragut et al., 2015a). A total of 255 samples were genotyped in this paper.
Subsequently, the same STR kit was used to genotype the Chueta population and 4 Jewish populations. In addition, in order to go further into the study of the X-chromosome, two other sets of markers (X-Alu and X-Indels) were also studied in these populations and Majorca (Ferragut et al., 2015b) (Ferragut et al., submitted). This work comprised 53 X-chromosome markers and a total of 500 samples genotyped. With this study, we aim not only to characterize and compare these six populations, but also to contrast the behaviour of these different X-chromosome markers in terms of Forensic and Population Genetics.
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Genetic analysis of 12 X-chromosome STRs in Western Mediterranean populations Ferragut JF a; Bentayebi Ka,b; Castro JAa; Ramon C a and Picornell Aa*
aInstitut Universitari d’Investigació en Ciències de la Salut (IUNICS) i Laboratori de Genètica, Departament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa, km 7.5, 07122, Palma de Mallorca, Illes Balears, Spain.
bBiochemistry and Immunology laboratory-Forensic Unit, Mohammed V Agdal University, Faculty of Sciences, Avenue Ibn Batouta Agdal, BP 1014 Rabat, Morocco
Abstract
Haplotype and allele frequencies of 12 X-STRs included in the Investigator Argus X-12 kit are reported for 255 individuals, representing four Western Mediterranean populations: Valencia (eastern mainland Spain) and the Balearic Islands (Majorca, Minorca and Ibiza). Ibiza shows the lowest intra-population variability and the highest level of linkage disequilibrium together with an important genetic distance with regard to the geographically close populations, which is consistent with the historical evidence for long-term demographic isolation and its different matrilineal background.
Key words: X-STRs, X-chromosome, Balearic Islands, Valencia, Western Mediterranean, Investigator Argus X-12 kit
*Corresponding author: Dr. Antònia Picornell. Laboratori de Genètica, Departament de Biologia, Universitat de les Illes Balears, Cra. Valldemossa, km 7.5, 07122-Palma de Mallorca, Illes Balears, Spain. Tel: +34-971-172050. Fax: +34-971-173184. E-mail:
apicornell@uib.es
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The Balearic archipelago (Majorca, Minorca and Ibiza islands, in the Western Mediterranean Sea) has been inhabited for 5500 years, and different people – especially the Romans (3rd century BC) and Catalans (early 13th century) – contributed to the genetic pool of the current population, but no remarkable additional contribution of foreign genes was received until recently because of immigration. Genetic studies (Rodríguez et al., 2009; Tomàs et al., 2006) show differences between the three islands and a remarkable differentiation of Ibiza compared to other Western Mediterranean populations, probably because of the Phoenician–Carthaginian origin of the first settlers and the effect of genetic drift. Few data regarding X-chromosome STRs have been reported in Western Mediterranean populations (Barbaro et al., 2012; Bentayebi et al., 2012) and there are no available data in the Balearic Islands. It is essential to give more insight into the genetic substructure in this area, focusing on known isolates such as the Ibiza population. In this work we studied the genetic diversity, geographic distribution and population structure of three insular populations from the Balearic Archipelago, and Valencia (eastern coast of the Iberian Peninsula), as a mainland Spanish reference.
Mouthwash samples were obtained from 255 unrelated individuals (160 males and 95 females), after informed consent. DNA was extracted by standard phenol-chloroform method. PCR amplification, capillary electrophoresis, data analysis and calculation of allele frequencies and additional relevant population and forensic statistical parameters were performed as previously described (Bentayebi et al., 2012). To examine the relationship of these populations with neighbouring populations, FST genetic distances, calculated using POPTREE2 (Takezaki et al., 2010), were performed to generate the multi-dimensional scaling (MDS) plot conducted with SPSS v.15.0 (SPSS, Inc., Chicago, IL, USA).
Table 1. Distribution of X-STR haplotypes for the linkage groups in 160 males from Balearic Islands (n=121) and Valencian (n=39) populations. observed heterozygosities ranging between 0.864 and 0.950. The lowest heterozygosity (0.480) was observed for the DXS7423 system in Minorca. No deviations from HWE
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were observed after Bonferroni correction. Typing of the 160 males from the Western Mediterranean region resulted in 160 different haplotypes when all 12 X-STRs were included. Linkage groups 1-4 revealed 135, 98, 93 and 103 haplotypes, respectively (Table 1). Among all the observed haplotypes, 96.4% showed frequencies <0.020 and the three most common haplotypes were observed in 8 individuals each, displaying a frequency of 0.050. In these populations, linkage group 1 proved to be the most polymorphic group and linkage group 3 the least, in accordance with other studies (Tomàs et al., 2012). The Valencia population showed greater haplotype diversity than Balearic Island populations, with a higher percentage of unique haplotypes in the four linkage groups. Supplementary Table 2 shows haplotype frequencies for each population. The Ibiza population showed the lowest haplotype diversity (HD) values – with a low percentage of unique haplotypes (between 46.3% and 56.1%) compared to the other populations studied (64.1% - 94.9%) – and the highest level of linkage disequilibrium (LD) in most of the loci pairs inside each linkage group, although after Bonferroni correction only one significant p-value was observed for DXS10148-DXS10135 pair of loci (p=0.000) (Supplementary Table 3). LD does not depend exclusively on the physical distance between loci, but may result from random genetic drift, founder effect, mutations, selection and population admixture or stratification (Chakravarti, 1999);
therefore the greater LD found in Ibiza could result from the founder effect and genetic drift related to the demographic and historical features of this population (an isolated, consanguineous population with a reduced effective population size), supporting previous genetic studies (Tomàs et al., 2006). When Balearic Island populations were pooled, significant LD was observed inside each linkage group. Although no significant differences in allelic frequency were observed in the Balearic populations for the 12 X-STRs studied, different studies emphasize the importance of investigating these populations and attempt to describe their roots and genetic substructure (Rodríguez et al., 2009; López‐ Escribano et al., 2013); therefore, these LD values could result from the heterogeneous distribution of the haplotypes among the islands. Forensic parameters of interest were calculated for each X-STR and population (Supplementary Table 1). The combined power of discrimination (PD) in females ranged from 1 in 4.74E+14 (in Ibiza) to 1 in 1.80E+15 (in Mallorca and Valencia). Combined PD males values ranged from 1 in 5.33E+08 (in Ibiza) to 1 in 1.04E+09 (in Valencia). A high combined MEC was also obtained for father-daughter duos and father-girl-mother trios (>0.99999 in all cases).
Although values differed slightly between populations, the set of loci in the Argus X-12 kit was highly informative in all the Western Mediterranean populations studied. A comparison between the Western Mediterranean population and 12 other European and African (mainly North African) populations with available data for the same set of X-STRS markers was performed. Supplementary Figure 1 shows a multi-dimensional scaling plot based on pairwise FST genetic distances. Along the X-axis, the distribution of the populations ranging from Northeast Africa to Northern Europe – with the Mediterranean populations showing an intermediate position – can be observed, as found in X-chromosome SNPs studies (Tomàs et al., 2008). However Majorca, Menorca and Valencia are closer to other European populations than to North Africans, in accordance with other studies that suggest the existence of a relative north-south gene flow barrier in
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the Western part of the Mediterranean area (Comas et al., 2000; Rodríguez et al., 2009).
Ibiza and Sahrawi populations show a remote position versus their geographic neighbouring populations; which may be due to historical and cultural background in the Sahrawi case. Regarding Ibiza, its differentiation versus other European populations was also evidenced by mtDNA analysis, but not by Y-chromosome markers, indicating a sex-biased contribution to the genetic pool of Ibiza (Tomàs et al., 2006). Indeed, as two thirds of the X-chromosomes descend from maternal origin, X-chromosome polymorphisms will mostly behave as matrilineal markers showing similar results to those obtained from mtDNA data. Therefore, the observed displaced plot of Ibiza based on X-STR results may reflect the differential matrilineal background of this population, probably accentuated by the effect of genetic drift. In conclusion, the results of the present study provide a useful X-STR database for the Western Mediterranean region. It is important to highlight that a local haplotype database must be used especially in Ibiza due to the matrilineal genetic features of this population.
Acknowledgments
This work was partially supported by grant AAEE133/09 from the Direcció General de R+D+I (Comunitat autònoma de les Illes Balears) and European Regional Development Fund (ERDF).
Supplementary Table 1. Allele frequencies of 12 X-STR in 350 chromosomes (Majorca (MAJ): 41 men and 22 women; Minorca (MIN): 39 men and 25 women; Ibiza (IBI): 41 males and 20 women; Valencia (VAL): 39 men and 28 women).
DXS10079 DXS10101 DXS10103 DXS10134 DXS10146 DXS10148
N 85 89 81 265 95 85 89 81 265 95 85 89 81 265 95 85 89 81 265 95 85 89 81 265 95 85 89 81 265 95
33 0.03 0.022 0.058 0.036 0.07 0.031 0.013 0.017 0.02 0.032
DXS7132 DXS7423 DXS8378 DXS10074 DXS10135 HPRTB
30.2
31 0.023 0.013 0.033 0.023 0.009
31.2
32 0.023 0.035 0.016 0.026 0.023
32.2
33 0.008 0.003 0.023
33.2
Het 0.734 0.747 0.729 0.741 0.712 0.696 0.685 0.708 0.696 0.698 0.696 0.684 0.706 0.704 0.705 0.840 0.835 0.824 0.839 0.855 0.923 0.924 0.909 0.933 0.927 0.743 0.728 0.716 0.731 0.751 Obs Het* 0.591 0.760 0.700 0.687 0.857 0.727 0.480 0.850 0.672 0.679 0.636 0.640 0.650 0.642 0.643 0.955 0.840 0.800 0.866 0.821 0.864 0.920 0.950 0.910 0.929 0.727 0.840 0.750 0.776 0.857 HW p-value* 0.528 0.333 0.782 0.473 0.264 0.644 0.034 0.115 0.021 0.226 0.673 0.274 0.754 0.143 0.865 0.021 0.977 0.752 0.238 0.795 0.114 0.352 0.439 0.013 0.941 0.663 0.773 0.666 0.859 0.530 PIC 0.689 0.704 0.679 0.697 0.663 0.646 0.631 0.660 0.645 0.644 0.635 0.621 0.653 0.646 0.647 0.820 0.814 0.801 0.819 0.838 0.918 0.919 0.902 0.928 0.923 0.700 0.684 0.668 0.686 0.712 PD female 0.884 0.893 0.877 0.889 0.868 0.858 0.847 0.866 0.857 0.854 0.847 0.837 0.860 0.854 0.855 0.954 0.952 0.946 0.954 0.962 0.989 0.989 0.985 0.991 0.990 0.891 0.882 0.871 0.883 0.899 PD male 0.734 0.747 0.729 0.741 0.712 0.696 0.685 0.708 0.696 0.698 0.696 0.684 0.706 0.704 0.705 0.840 0.835 0.824 0.839 0.855 0.923 0.924 0.909 0.933 0.927 0.743 0.728 0.716 0.731 0.751 PE 0.483 0.504 0.475 0.494 0.447 0.422 0.405 0.441 0.422 0.426 0.422 0.404 0.438 0.434 0.436 0.675 0.666 0.644 0.674 0.704 0.843 0.844 0.813 0.862 0.852 0.497 0.472 0.454 0.478 0.512 PI 1.879 1.975 1.848 1.930 1.737 1.644 1.585 1.714 1.645 1.658 1.645 1.583 1.702 1.687 1.695 3.124 3.031 2.840 3.112 3.445 6.492 6.555 5.466 7.410 6.880 15.102 13.475 12.408 13.820 16.153 MEC Desmarais 0.689 0.704 0.679 0.697 0.663 0.646 0.631 0.660 0.645 0.644 0.635 0.621 0.653 0.646 0.647 0.820 0.814 0.801 0.819 0.838 0.918 0.919 0.902 0.928 0.923 0.700 0.684 0.668 0.686 0.712 MEC Desmarais duo 0.548 0.566 0.536 0.556 0.520 0.502 0.486 0.517 0.501 0.499 0.490 0.474 0.508 0.501 0.503 0.708 0.702 0.685 0.708 0.733 0.853 0.855 0.828 0.871 0.861 0.560 0.544 0.526 0.546 0.574
* Calculated using female data.
BI– pooled Balearic Islands; N– Number of X-chromosomes studied; Het– expected heterozygosity; Obs Het– observed heterozygosity; HW– Hardy-Weinberg; PIC – polymorphic information content; PDfemale – power of discrimination in women; PDmale – power of discrimination in men; PE- power of exclusion; PI- paternity Index; MEC Desmarais– mean exclusion chance in trios involving daughter; MEC Desmarais duo – mean exclusion chance in father/daughter or mother/son duos.
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Supplementary Table 2. X-chromosome haplotypes of four linked STR trios in 121 males from Balearic Islands (41 from Majorca, 39 from Minorca and 41 from Ibiza) and 39 males from Valencia populations. More common haplotypes (5%) are labelled with *.
Linkage group 1
DXS10148 DXS10135 DXS8378 Majorca Minorca Ibiza Valencia DXS10148 DXS10135 DXS8378 Majorca Minorca Ibiza Valencia
18 18 11 1 24.1 31 10 1
Haplotype diversity ± Majorca Minorca Ibiza Valencia
standard desviation 0.9951 ± 0.0072 0.9973 ± 0.0066 0.9866 ± 0.0086 1.0000 ± 0.0058
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Linkage group 2
DXS7132 DXS10079 DXS10074 Majorca Minorca Ibiza Valencia DXS7132 DXS10079 DXS10074 Majorca Minorca Ibiza Valencia
11 24 16 1 14 18 15 1
Haplotype diversity ± Majorca Minorca Ibiza Valencia
standard desviation 0.9915 ± 0.0077 0.9852 ± 0.0104 0.9841 ± 0.0087 0.9960 ± 0.0069
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Linkage group 3
DXS10103 HPRTB DXS10101 Majorca Minorca Ibiza Valencia DXS10103 HPRTB DXS10101 Majorca Minorca Ibiza Valencia
15 12 33 1 19 11 28.2 1
Haplotype diversity ± Majorca Minorca Ibiza Valencia
standard desviation 0.9866 ± 0.0095 0.9879 ± 0.0087 0.9829 ± 0.0088 0.9919 ± 0.0082
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Linkage group 4
DXS10146 DXS10134 DXS7423 Mallorca Menorca Eivissa València DXS10146 DXS10134 DXS7423 Mallorca Menorca Eivissa València
24 37 14 1 29 34 15 1
Haplotype diversity ± Majorca Minorca Ibiza Valencia
standard desviation 0.9963 ± 0.0063 0.9852 ± 0.0104 0.9866 ± 0.0081 0.9919 ± 0.0082
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Supplementary Table 3. Linkage disequilibrium inside the 4 Linkage Groups (LG) in Majorca, Minorca, Ibiza and Valencia populations. In bold, significant p-values after Bonferroni correction.
pair locus Majorca Minorca Ibiza Balearic Islands (pooled) Valencia
(DXS10148, DXS10135) 0.1890 0.1492 0.0000 0.0000 0.9338
(DXS10148, DXS8378) 0.5582 0.7408 0.0029 0.2295 0.8233
(DXS10135, DXS8378) 0.2457 0.6266 0.0049 0.1099 0.6107
(DXS7132, DXS10079) 0.9933 0.0863 0.0413 0.3657 0.1456
(DXS7132, DXS100747) 0.1752 0.4016 0.0955 0.0392 0.1567
(DXS10079, DXS10074) 0.2440 0.0069 0.0067 0.0000 0.6035
(DXS10103, HPRTB) 0.1558 0.2716 0.1435 0.0271 0.4451
(DXS10103, DXS10101) 0.2360 0.0113 0.0606 0.0000 0.0079
(HPRTB, DXS10101) 0.4058 0.1254 0.0048 0.0000 0.0504
(DXS10146, DXS10134) 0.3456 0.4500 0.4618 0.0000 0.0156
(DXS10146, DXS7423) 0.0248 0.3892 0.0401 0.1418 0.0965
(DXS10134, DXS7423) 0.7415 0.0000 0.7737 0.7527 0.6525
Supplementary Figure 1. Multidimensional scaling analysis (MDS) based on Reynolds genetic distances calculated between populations. African populations are represented by circular points, European populations by square dots and Balearic and Valencia populations are labelled with stars.
Moroccan Berbers, Moroccan Arabs and Sahrawi (Bentayebi K et al. 2011. Forensic Sci Int Genet 6: e48-49); Denmark and Somalia (Tomàs C et al. 2012. Int J Legal Med 126:121-128); Sweden (Tillmar AO. 2012.
Forensic Sci Int Genet 6: e80-81); Portugal (Cainé L et al. 2013 Int J Legal Med 127:63-64); Hungary (Horváth G et al. 2012. Forensic Sci Int Genet 6: e46-47); Germany (Edelmann J et al.2012. Forensic Sci Int Genet 6:e24-34); Algeria (Bekada A et al. 2010. Int J Legal Med 124:287-294); Italy (Inturri S et al. 2011. Forensic Sci Int Genet 5:e152-154).
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Genetic diversity of 12 X-chromosomal short tandem repeats in Jewish populations J.F. Ferragut1, J.A. Castro1, C. Ramon1 and A. Picornell1
1Institut Universitari d'Investigacions en Ciències de la Salut (IUNICS) and Laboratori de Genètica, Departament de Biologia, Universitat de les Illes Balears. Palma de Mallorca, Spain
Corresponding author: Antonia Picornell (apicornell@uib.es). Cra. Valldemossa Km 7,5.
07122-Palma (Balearic Islands, SPAIN). Telephone number: +34-971172050. Fax number:
+34-971173184.
Abstract: Haplotype and allele frequencies of 12 X-STRs included in the Investigator Argus X-12 kit were studied for 313 individuals, representing five populations with known Jewish ancestry. The high efficiency in forensic parameters in all the populations studied demonstrates that this set of markers provides a powerful tool for solving complex kinship cases in Jewish populations.
Keywords: X-STRs; X-Chromosome; Investigator Argus X-12 kit; Jews; Chuetas.
106 1. Introduction
X-Short Tandem Repeat (X-STR) markers have a series of special characteristics that justify their increasing interest in forensic practice, population genetics, and anthropology.
Compared with autosomes, the X-chromosome has lower recombination and mutation rates and a smaller effective population size, resulting in faster genetic drift. Consequently, both linkage disequilibrium (LD) and population structure in the X-chromosome are expected to be stronger than in autosomes. X-STRs are particularly useful in paternity testing and kinship analysis, especially in deficient cases, such as grandmother-granddaughter, aunt-niece and cousins. Owing to the complexity of kinship investigation, a new approach of substituting single STRs for stable haplotypes of closely linked loci has been suggested.
Jews can be traced back to populations occupying a small geographic area, in the Middle East, several thousand years ago and have maintained continuous cultural and religious traditions despite a series of Diasporas. Contemporary Jews comprise several communities that can be classified according to the location where each community developed. Among others, these include Middle Eastern Jews (Mizrahim) (Iran and Iraq), who have always resided in the Middle East, dating from Babylonian or Persian communities in the fourth to sixth centuries BCE; the Askenazim – the vast majority of living Jews – who lived in communities in central and eastern Europe, but whose origins remain highly contested and enigmatic to this day; the Sephardim ("Spanish" in Hebrew) who, after their expulsion from the Iberian Peninsula in the late 15th century, lived in other Mediterranean countries (especially Bulgaria and Turkey), where they mixed with local Jewish communities formed during classical antiquity; and North African Jews, comprising both Sephardim and Mizrahim, as there exists evidence of Jewish communities in North Africa as early as the first centuries AD that were augmented as a consequence of the Spanish expulsion (Ben-Sasson et al., 1976). Chuetas are an isolated, inbred Spanish community, descending from Majorcan Sephardic Jews. Their peculiar history has kept the memory of their Jewish origin and has prevented their gradual assimilation into the general population (Laub and Laub, 1987).
The present study analyzed 474 X-chromosomes from five populations with Jewish ancestry, aiming to build an X-STR database, based on the markers included in the Investigator Argus X-12 kit (Qiagen, Hilden, Germany), for anthropological and forensic purposes.
