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Haplotype analysis of α-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews
European Journal of Medical Genetics xxx (2016) 1e4
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews Adir Shaulov*, Dvora Filon, Deborah Rund Department of Hematology, Hadassah - Hebrew University Hospital, Jerusalem, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 June 2016 Accepted 16 October 2016 Available online xxx
a-Thalassemia (a-thal) is among the world's most common single gene disorders, generally attributed to a selective advantage of heterozygotes against malaria mortality. A high frequency of -a3.7 deletion
Keywords: Globin genes Selection pressure Thalassemia Gene deletions Ashkenazi jews
heterozygosity has been previously reported in Ashkenazi Jews despite lack of obvious malarial selection pressure in this population. Using haplotype and -a3.7 subtype analysis we analyzed a subset of -a3.7 homozygotes from various Israeli ethnic groups. We found a high frequency of the common Ia haplotype in Yemenite Jews and Arabs (54% and 13% respectively). Ashkenazi Jews exhibited a high frequency of IIIb alleles (67%) previously reported only in Aboriginal Australians and not found in other Israeli ethnicities. Both Yemenites and Ashkenazim carried the rare IIh alleles (18% and 15% respectively). These results may suggest multiple founder effects in Ashkenazi Jews as well a common founder for both Yemenite and Ashkenazi Jews. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
a-Thalassemia (a-thal) is among the world's most common single gene disorders and is the result of an imbalance in the production of alpha and b-globin chains of the hemoglobin molecule (Piel and Weatherall, 2014). Most cases of a-thalassemia are a result of inheriting deletions of one or more of the normal complement of 4 a-globin genes. These deletions occurred as a result of events of unequal crossing over between homologous regions of the a-globin genes. The paired a-globin genes are located on the short arm of chromosome 16 within the a-globin gene cluster, which also contains an embryonic gene and several homologous pseudogenes (Higgs et al., 1989). This locus contains multiple polymorphic sites exhibiting genetic linkage, forming distinct haplotypes which are found with a variable distribution in widespread geographical areas (Table 1) (Higgs et al., 1986). At the hematological level, alpha thalassemia trait results in variable degrees of anemia and microcytosis (Piel and Weatherall, 2014) (small size of red blood cells which is measured by
Abbreviations: thal, Thalassemia. * Corresponding author. Department of Hematology, Hadassah - Hebrew University Hospital, Ein Kerem, POB 12000, Jerusalem, 91120, Israel. E-mail address: [email protected] (A. Shaulov).
automated blood counting). The most common of a-thalassemia alleles is the result of the deletion of a 3.7 kilo-base region from one chromosome, -a3.7, with a reciprocal addition to the opposite chromosome, aaaanti 3.7. -a3.7 chromosomes can be further subdivided into three separate subgroups based on polymorphic sites, each correlating with a distinct crossing over event (Fig. 1) (Higgs et al., 1984) which occurred as separate genetic events. The short arm of chromosome 16 includes the alpha-2 gene followed by the alpha-1 gene. Alpha-1 and alpha-2 include three homologous regions named X, Y and Z. Pairing between homologous regions and resultant unequal crossing over results in the commonly found -a3.7 deletion. The specific points of unequal crossing over result in three distinct subtypes of the -a3.7 deletion; I, II and III, identified by the inclusion or exclusion of restriction sites for ApaI and RsaI. The prevalence of the -a3.7 chromosome is many fold greater than that of the aaaanti 3.7 chromosome, suggesting positive selection for the -a3.7 allele. In addition, the geographic distribution of a-thalassemia demonstrates prevalence in Africa, Southeast Asia and the Mediterranean, which has been generally attributed to a survival advantage during infection with the intra-erythrocytic falciparum malaria parasite (Flint et al., 1986). While the precise mechanism of this selective advantage is not known, epidemiological evidence strongly supports its existence (Kwiatkowski, 2005) (Sakai et al., 2000). In Israel, the ethnic diversity of the population is mirrored in the
http://dx.doi.org/10.1016/j.ejmg.2016.10.008 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
2
A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
Table 1 Haplotypes found in Israeli ethnic groups and reported worldwide distribution. Haplotype
Ia Ib IIa IIc IIg IIh IIIa IIIb IIIe VIa
Polymorphic sites
Distribution (Sakai et al., 2000) (Roberts-Thomson et al., 1996) (Martinson et al., 1995) (Hertzberg et al., 1988)
XbaI
SacI
BglI
SML
PZ/Z
AccI
RsaI
PstI*
PstI
Europe
Asia
Polynesia
Senegal
Central Australia
þ e e þ þ e e þ e e
þ þ þ þ e e e e þ þ
e e e e e þ þ þ e e
M M L M M M M M M L
PZ PZ PZ PZ PZ PZ Z Z Z PZ
þ þ þ þ þ þ e e e e
þ þ e e e e e e e e
e e e e e e e e e e
e e e e e e e e e e
62% 2% 14% 1% e e 8% 7% 1% 1%
41% 2% 23% 3% 2% e 7% 1% e 1%
20% e 7% 3% 1% e 8% 1% e e
11% 2% e 18% 4% e 1% 1% 1% e
<1% e e e e 2% 73% <1% e e
*Restriction site not present in -a3.7 chromosomes.
Fig. 1. a3.7 subtypes.
wide variability of a-globin mutations (Oron-Karni et al., 2000). In previous research from our laboratory we evaluated 232 individuals with undiagnosed microcytic anemia. 303 of 464 chromosomes evaluated were found to carry a-globin mutations (OronKarni et al., 2000). Surprisingly, despite the fact that Europe is not endemic for malaria, 17% of chromosomes carrying a-globin mutations were of Ashkenazi origin. In an additional study we have found the -a3.7 allele at a carrier frequency of 7.9% and allele frequency of 0.04% among Ashkenazi Jews (Rund et al., 2004). The
prevalence of a-globin mutations in Ashkenazim was similar to that of Yemenite Jews whose geographic origin was one with continuous exposure to malaria. In both populations, mutations of the aglobin gene included mostly -a3.7 chromosomes, in contrast with other Israeli ethnic groups in which other deletions and point mutations of the a-globin gene were more common (Rund et al., 2004). In addition, other hemoglobinopathies such as sickle cell anemia and b-thalassemia are rare in Ashkenazim (Oppenheim et al., 1993) while a higher rate might be expected if malarial
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
selective pressure existed in the geographic regions in which they originated. This suggests that founder effect may be the primary reason for the high prevalence of -a3.7 chromosomes in Ashkenazi Jews. In this study we aimed to elucidate the genetic mechanism(s) responsible for the high frequency of a-thal in Ashkenazi Jews, based on genetic analysis of the a-globin gene cluster in this ethnic group including analysis of -a3.7 subtype and haplotypes. We hypothesized that we would be able to determine if a founder effect would explain the frequency, as has been found with other genetic diseases common in this ethnic group (Motulsky, 1995). We also sought, if possible, to clarify the origin of the deletion in this ethnic group by comparing the genetic analysis of Ashkenazim to that of other ethnic groups in our population. 2. Material and methods For over 20 years, our laboratory has been a referral center for DNA based analysis of thalassemia patients from all over Israel. As such we performed globin gene analysis on several hundred individuals found to have microcytic anemia who were not carriers of beta thalassemia trait using hemoglobin electrophoresis or DNA sequencing. This was the referral patient population for this study. The Institutional Helsinki Committee gave its approval for this research. DNA was isolated from peripheral blood leukocytes according to standard procedures (Goossens and Kan, 1981). PCR was performed for the -a3.7 allele as previously described (Oron-Karni et al., 1998). We selected all samples of individuals found to be homozygous for the -a3.7 allele. We reasoned that homozygotes would be a genetically homogeneous group which would facilitate haplotyping. Using PCR and restriction enzyme analysis with ApaI and RsaI according to published protocols (Higgs et al., 1984), we determined the specific 3.7 kb deletion subtype (Fig. 1). We then performed haplotype analysis of the a-globin gene locus based on 9 distinct published polymorphic sites (Miles et al., 2001), 8 of which are present on the -a3.7 chromosome. 3. Results We evaluated 174 chromosomes of 87 -a3.7 homozygotes from several ethnic groups: Ashkenazi Jews: 55 chromosomes, Yemenite Jews: 54, Iraqi Jews: 11, Arabs: 28, Druze 6 and other Sephardi Jews: 14. Ethnicity was self-declared, and only seven individuals declared mixed ethnicity. Using PCR and digestion with ApaI and RsaI, it was determined that 163 of 170 (96%) chromosomes are of the -a3.7I type. An additional 4 alleles were of the -a3.7II type; one Yemenite and 2 Ashkenazi -a3.7I/-a3.7II heterozygotes and one Arab -a3.7II/-a3.7II homozygote. Two samples showed heterozygosity for both ApaI and RsaI restriction, and -a3.7 subtype could not be elucidated, possibly due to an additional crossing over event (Law et al., 2006).
3
Full haplotype restriction pattern was elucidated for 82 samples (94%). In 5 Ashkenazi samples, 1 Yemenite and 1 sample of other origin a full restriction pattern could not be achieved due to technical difficulties. Samples with homozygous haplotypes could be directly identified using restriction sites (Table 2). Two distinct haplotypes could be identified in samples with single restriction site heterozygosity. 73/174 (42%) of chromosomes could thus be identified showing 12 different haplotypes (Ia, Ib, IIa, IIc, IIg, IIh, IIIa, IIIb, IIIe, VIa and 2 newly reported haplotypes). Among Ashkenazim 9 IIIb homozygotes and one IIIb/IIIa compound heterozygote were identified. In total 19/55 (35%) of Ashkenazi chromosomes carried haplotype IIIb. Other than 1 Iraqi homozygote, no other ethnicities exhibited IIIb haplotype. 8 Ia homozygotes and 5 heterozygotes were found in the Yemenite population. In addition 3 IIh homozygotes were found. Among Arabs; 5 Ia homozygotes and one heterozygote were found. 42% of Yemenite chromosomes and 39% of Arab chromosomes carried the Ia haplotype. Based on the high prevalence of Ia in Yemenites and Arabs and IIIb in Ashkenazim we analyzed the remaining samples with multiple restriction site heterozygosity assuming that the prevalent haplotype was present on at least one chromosome (Table 3). Using this analysis 54% of Yemenite and 46% of Arab chromosomes carried the Ia haplotype whereas 67% of Ashkenazi chromosomes carried the IIIb haplotype. In addition to the three Yemenite IIh homozygotes, reanalysis showed 6 probable IIIb/IIh heterozygotes among Ashkenazim and an additional 2 samples that may be heterozygous for IIh. The unavailability of DNA of parents and/or other family members precluded definitive characterization. 4. Discussion In this study we sought to compare alpha globin haplotypes of a group of 87 individuals who were homozygous for the highly prevalent -a3.7 deletion. These individuals were of various ethnicities, primarily Ashkenazi (32%) and Yemenite (31%) Jews. -a3.7 subtyping showed an expected uniform picture with 96% of chromosomes being of the -a3.7I type most commonly found worldwide. Unexpectedly, at the level of the haplotype, a great degree of diversity was found: at least 10 different haplotypes were deduced among Ashkenazim with a large number of chromosomes carrying unknown haplotypes. Interestingly, two thirds of Ashkenazi
Table 3 Common haplotypes and distribution in studied -a3.7 homozygotes following reanalysis based on dominant haplotype. Alleles
Ashkenazim (n ¼ 55)
Yemenites (n ¼ 50)
Arabs (n ¼ 28)
IA IIIB IIH Other
0 37 (67%) 8 (15%) 10 (18%)
27 (54%) 0 (0%) 9 (18%) 14 (28%)
13 (46%) 0 (0%) 0 (0%) 15 (54%)
Table 2 Haplotypes and Distribution in studied -a3.7 homozygotes. Alleles
Ashkenazi Jews (n ¼ 55)
Yemenite Jews (n ¼ 50)
Arab (n ¼ 28)
Iraqi Jews (n ¼ 11)
Druze (n ¼ 6)
Other Jews (n ¼ 24)
Total (n ¼ 174)
IA IIIB IIH Other alleles
0 19 (35%) 0 IIIA e 1
21 (42%) 0 6 (12%) IB e 1 IIC e 3 IIA e 1 IIB e 1 Unknown e 1
11 (39%) 0 e IIC e 1
1 (3%) 2 (18%) e IIIE e 2
0 0 e IIC e 1
1 (4%) 2 (8%) e IIG e 1
33 (19%) 21 (12%) e
IIA e 1 VIA e 1
Unknown e 1
IIA e 2 IIIA e 2
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
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A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
chromosomes carried haplotype IIIb which is common in Melanesia and Papua New Guinea but rarely found in Europe and Saudi Arabia (Flint et al., 1992) (Table 1). Its occurrence in a European ethnic group is not likely to have occurred by migration and more likely occurred by recurrent mutational events at multiple sites. In contrast, only 1.7% of non-Ashkenazi chromosomes carried haplotype IIIb. Nearly half of chromosomes from Arabs and Yemenite Jews carried haplotype Ia. Iraqi Jews and other Sephardi Jewish ethnic groups showed a lower prevalence of Ia while no Ashkenazi samples carried the Ia haplotype. Ia is the most common haplotype globally and the lack of prevalence in Ashkenazim coupled with the high prevalence of the rare haplotype IIIB may provide support for founder effect and genetic drift in Ashkenazim as is the case in other diseases such as Factor XI deficiency, Gaucher, Tay Sachs and others (Motulsky, 1995). Interestingly 3 homozygotes for haplotype IIh were identified in Yemenite Jews, a haplotype previously identified in Aboriginal Australians (Roberts-Thomson et al., 1996). Analysis of results, as described above, based on prevalence of the haplotype common in the ethnic group e Ia in Yemenites and IIIa in Ashkenazim surprisingly identified an additional 3 IIh chromosomes in Yemenites (total prevalence - 18%), and 8 IIh chromosomes in Ashkenazim (prevalence - 15%). That such a rare haplotype may be found in two distinct Jewish ethnic groups which were geographically separated for centuries may well suggest that the IIh is a remnant of a very early founder common to both Ashkenazi and Yemenite Jews. The origin of Ashkenazi Jews has been researched extensively using various genetic tools such as DNA sequencing (Carmi et al., 2014), Y chromosome analysis (Nebel et al., 2005) and mitochondrial DNA (Costa et al., 2013). These have shown a founder effect with Near East genetic imprint as well as variation in genetic markers, as influenced by local populations. Our study of Ashkenazim is unique in that we began by selecting a group of individuals for analysis in whom the genetic evidence pointed to a Middle Eastern origin as evidenced by homozygous -a3.7. We previously showed evidence of high prevalence of the -a3.7 deletion in Ashkenazim which is not prevalent in European populations and yet carries a haplotype distinct from that of other Jewish ethnic groups. The common haplotype, IIh, is distinct from Ia in 4/8 polymorphic sites while IIIa is distinct from IIh in only 2/8. Ia and IIIa differ in 5/8 sites. IIIa may be seen as a result of genetic drift of the common IIh haplotype while Ia is the result of natural selection and the genetic influence of surrounding populations. In summary, haplotype analysis of a homogeneous group of -a3.7 deletion homozygotes showed a dominant and rare IIIa haplotype in addition to a significant population of IIh haplotype chromosomes which may originate from a founder common to other Jewish ethnicities. Haplotype analysis may provide pertinent information about the prevalence of alpha-thalassemia in areas not endemic to malaria. Acknowledgements We thank the Rochlin foundation for their long term support for our research in thalassemia. References
Avraham, D., Mukherjee, S., Bowen, B.M., Thomas, T., Vijai, J., Cruts, M., Froyen, G., Lambrechts, D., Plaisance, S., Van Broeckhoven, C., Van Damme, P., Van Marck, H., Barzilai, N., Darvasi, A., Offit, K., Bressman, S., Ozelius, L.J., Peter, I., Cho, J.H., Ostrer, H., Atzmon, G., Clark, L.N., Lencz, T., Pe’er, I., 2014. Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins. Nat. Commun. 5, 4835. http://dx.doi.org/10.1038/ncomms5835. Costa, M.D., Pereira, J.B., Pala, M., Fernandes, V., Olivieri, A., Achilli, A., Perego, U.A., Rychkov, S., Naumova, O., Hatina, J., Woodward, S.R., Eng, K.K., Macaulay, V., Carr, M., Soares, P., Pereira, L., Richards, M.B., 2013. A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages. Nat. Commun. 4, 2543. http://dx.doi.org/10.1038/ncomms3543. Flint, J., Hill, A.V., Bowden, D.K., Oppenheimer, S.J., Sill, P.R., Serjeantson, S.W., BanaKoiri, J., Bhatia, K., Alpers, M.P., Boyce, A.J., 1986. High frequencies of alphathalassaemia are the result of natural selection by malaria. Nature 321, 744e750. http://dx.doi.org/10.1038/321744a0. Flint, J., Clegg, J.B., Boyce, A.J., 1992. Molecular Genetics of Globin Genes and Human Population Structure. in: Mol. Appl. Biol. Anthropol.. Cambridge University Press, pp. 103e178. Goossens, M., Kan, Y.Y., 1981. DNA analysis in the diagnosis of hemoglobin disorders. Methods Enzymol. 76, 805e817. Hertzberg, M.S., Mickleson, K.N., Trent, R.J., 1988. Alpha-globin gene haplotypes in Polynesians: their relationships to population groups and gene rearrangements. Am. J. Hum. Genet. 43, 971e977. Higgs, D.R., Hill, A.V., Bowden, D.K., Weatherall, D.J., Clegg, J.B., 1984. Independent recombination events between the duplicated human alpha globin genes; implications for their concerted evolution. Nucleic Acids Res. 12, 6965e6977. Higgs, D.R., Wainscoat, J.S., Flint, J., Hill, A.V., Thein, S.L., Nicholls, R.D., Teal, H., Ayyub, H., Peto, T.E., Falusi, A.G., 1986. Analysis of the human alpha-globin gene cluster reveals a highly informative genetic locus. Proc. Natl. Acad. Sci. U. S. A. 83, 5165e5169. Higgs, D.R., Vickers, M.A., Wilkie, A.O., Pretorius, I.M., Jarman, A.P., Weatherall, D.J., 1989. A review of the molecular genetics of the human alpha-globin gene cluster. Blood 73, 1081e1104. Kwiatkowski, D.P., 2005. How malaria has affected the human genome and what human genetics can teach us about malaria. Am. J. Hum. Genet. 77, 171e192. http://dx.doi.org/10.1086/432519. Law, H.-Y., Luo, H.-Y., Wang, W., Ho, J.F.V., Najmabadi, H., Ng, I.S.L., Steinberg, M.H., Chui, D.H.K., Chong, S.S., 2006. Determining the cause of patchwork HBA1 and HBA2 genes: recurrent gene conversion or crossing over fixation events. Haematologica 91, 297e302. Martinson, J.J., Excoffier, L., Swinburn, C., Boyce, A.J., Harding, R.M., Langaney, A., Clegg, J.B., 1995. High diversity of alpha-globin haplotypes in a Senegalese population, including many previously unreported variants. Am. J. Hum. Genet. 57, 1186e1198. Miles, K.L., Norwich, J.T., Martinson, J.J., Clegg, J.B., 2001. Polymerase chain reaction protocols for alpha globin haplotype polymorphisms. Br. J. Haematol. 113, 694e698. Motulsky, A.G., 1995. Jewish diseases and origins. Nat. Genet. 9, 99e101. http:// dx.doi.org/10.1038/ng0295-99. Nebel, A., Filon, D., Faerman, M., Soodyall, H., Oppenheim, A., 2005. Y chromosome evidence for a founder effect in Ashkenazi Jews. Eur. J. Hum. Genet. EJHG 13, 388e391. http://dx.doi.org/10.1038/sj.ejhg.5201319. Oppenheim, A., Oron, V., Filon, D., Fearon, C.C., Rachmilewitz, E.A., Kazazian, H.H., Rund, D., 1993. Sporadic alleles, including a novel mutation, characterize betathalassemia in Ashkenazi Jews. Hum. Mutat. 2, 155e157. http://dx.doi.org/ 10.1002/humu.1380020219. Oron-Karni, V., Filon, D., Oppenheim, A., Rund, D., 1998. Rapid detection of the common Mediterranean alpha-globin deletions/rearrangements using PCR. Am. J. Hematol. 58, 306e310. Oron-Karni, V., Filon, D., Shifrin, Y., Fried, E., Pogrebijsky, G., Oppenheim, A., Rund, D., 2000. Diversity of alpha-globin mutations and clinical presentation of alpha-thalassemia in Israel. Am. J. Hematol. 65, 196e203. Piel, F.B., Weatherall, D.J., 2014. The a-Thalassemias, N. Engl. J. Med. 371, 1908e1916. http://dx.doi.org/10.1056/NEJMra1404415. Roberts-Thomson, J.M., Martinson, J.J., Norwich, J.T., Harding, R.M., Clegg, J.B., Boettcher, B., 1996. An ancient common origin of aboriginal Australians and New Guinea highlanders is supported by alpha-globin haplotype analysis. Am. J. Hum. Genet. 58, 1017e1024. Rund, D., Filon, D., Jackson, N., Asher, N., Oron-Karni, V., Sacha, T., Czekalska, S., Oppenheim, A., 2004. An unexpectedly high frequency of heterozygosity for alpha-thalassemia in Ashkenazi Jews. Blood Cells. Mol. Dis. 33, 1e3. http:// dx.doi.org/10.1016/j.bcmd.2004.04.009. Sakai, Y., Kobayashi, S., Shibata, H., Furuumi, H., Endo, T., Fucharoen, S., Hamano, S., Acharya, G.P., Kawasaki, T., Fukumaki, Y., 2000. Molecular analysis of alphathalassemia in Nepal: correlation with malaria endemicity. J. Hum. Genet. 45, 127e132. http://dx.doi.org/10.1007/s100380050198.
Carmi, S., Hui, K.Y., Kochav, E., Liu, X., Xue, J., Grady, F., Guha, S., Upadhyay, K., Ben-
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews Adir Shaulov*, Dvora Filon, Deborah Rund Department of Hematology, Hadassah - Hebrew University Hospital, Jerusalem, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 June 2016 Accepted 16 October 2016 Available online xxx
a-Thalassemia (a-thal) is among the world's most common single gene disorders, generally attributed to a selective advantage of heterozygotes against malaria mortality. A high frequency of -a3.7 deletion
Keywords: Globin genes Selection pressure Thalassemia Gene deletions Ashkenazi jews
heterozygosity has been previously reported in Ashkenazi Jews despite lack of obvious malarial selection pressure in this population. Using haplotype and -a3.7 subtype analysis we analyzed a subset of -a3.7 homozygotes from various Israeli ethnic groups. We found a high frequency of the common Ia haplotype in Yemenite Jews and Arabs (54% and 13% respectively). Ashkenazi Jews exhibited a high frequency of IIIb alleles (67%) previously reported only in Aboriginal Australians and not found in other Israeli ethnicities. Both Yemenites and Ashkenazim carried the rare IIh alleles (18% and 15% respectively). These results may suggest multiple founder effects in Ashkenazi Jews as well a common founder for both Yemenite and Ashkenazi Jews. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
a-Thalassemia (a-thal) is among the world's most common single gene disorders and is the result of an imbalance in the production of alpha and b-globin chains of the hemoglobin molecule (Piel and Weatherall, 2014). Most cases of a-thalassemia are a result of inheriting deletions of one or more of the normal complement of 4 a-globin genes. These deletions occurred as a result of events of unequal crossing over between homologous regions of the a-globin genes. The paired a-globin genes are located on the short arm of chromosome 16 within the a-globin gene cluster, which also contains an embryonic gene and several homologous pseudogenes (Higgs et al., 1989). This locus contains multiple polymorphic sites exhibiting genetic linkage, forming distinct haplotypes which are found with a variable distribution in widespread geographical areas (Table 1) (Higgs et al., 1986). At the hematological level, alpha thalassemia trait results in variable degrees of anemia and microcytosis (Piel and Weatherall, 2014) (small size of red blood cells which is measured by
Abbreviations: thal, Thalassemia. * Corresponding author. Department of Hematology, Hadassah - Hebrew University Hospital, Ein Kerem, POB 12000, Jerusalem, 91120, Israel. E-mail address: [email protected] (A. Shaulov).
automated blood counting). The most common of a-thalassemia alleles is the result of the deletion of a 3.7 kilo-base region from one chromosome, -a3.7, with a reciprocal addition to the opposite chromosome, aaaanti 3.7. -a3.7 chromosomes can be further subdivided into three separate subgroups based on polymorphic sites, each correlating with a distinct crossing over event (Fig. 1) (Higgs et al., 1984) which occurred as separate genetic events. The short arm of chromosome 16 includes the alpha-2 gene followed by the alpha-1 gene. Alpha-1 and alpha-2 include three homologous regions named X, Y and Z. Pairing between homologous regions and resultant unequal crossing over results in the commonly found -a3.7 deletion. The specific points of unequal crossing over result in three distinct subtypes of the -a3.7 deletion; I, II and III, identified by the inclusion or exclusion of restriction sites for ApaI and RsaI. The prevalence of the -a3.7 chromosome is many fold greater than that of the aaaanti 3.7 chromosome, suggesting positive selection for the -a3.7 allele. In addition, the geographic distribution of a-thalassemia demonstrates prevalence in Africa, Southeast Asia and the Mediterranean, which has been generally attributed to a survival advantage during infection with the intra-erythrocytic falciparum malaria parasite (Flint et al., 1986). While the precise mechanism of this selective advantage is not known, epidemiological evidence strongly supports its existence (Kwiatkowski, 2005) (Sakai et al., 2000). In Israel, the ethnic diversity of the population is mirrored in the
http://dx.doi.org/10.1016/j.ejmg.2016.10.008 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
2
A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
Table 1 Haplotypes found in Israeli ethnic groups and reported worldwide distribution. Haplotype
Ia Ib IIa IIc IIg IIh IIIa IIIb IIIe VIa
Polymorphic sites
Distribution (Sakai et al., 2000) (Roberts-Thomson et al., 1996) (Martinson et al., 1995) (Hertzberg et al., 1988)
XbaI
SacI
BglI
SML
PZ/Z
AccI
RsaI
PstI*
PstI
Europe
Asia
Polynesia
Senegal
Central Australia
þ e e þ þ e e þ e e
þ þ þ þ e e e e þ þ
e e e e e þ þ þ e e
M M L M M M M M M L
PZ PZ PZ PZ PZ PZ Z Z Z PZ
þ þ þ þ þ þ e e e e
þ þ e e e e e e e e
e e e e e e e e e e
e e e e e e e e e e
62% 2% 14% 1% e e 8% 7% 1% 1%
41% 2% 23% 3% 2% e 7% 1% e 1%
20% e 7% 3% 1% e 8% 1% e e
11% 2% e 18% 4% e 1% 1% 1% e
<1% e e e e 2% 73% <1% e e
*Restriction site not present in -a3.7 chromosomes.
Fig. 1. a3.7 subtypes.
wide variability of a-globin mutations (Oron-Karni et al., 2000). In previous research from our laboratory we evaluated 232 individuals with undiagnosed microcytic anemia. 303 of 464 chromosomes evaluated were found to carry a-globin mutations (OronKarni et al., 2000). Surprisingly, despite the fact that Europe is not endemic for malaria, 17% of chromosomes carrying a-globin mutations were of Ashkenazi origin. In an additional study we have found the -a3.7 allele at a carrier frequency of 7.9% and allele frequency of 0.04% among Ashkenazi Jews (Rund et al., 2004). The
prevalence of a-globin mutations in Ashkenazim was similar to that of Yemenite Jews whose geographic origin was one with continuous exposure to malaria. In both populations, mutations of the aglobin gene included mostly -a3.7 chromosomes, in contrast with other Israeli ethnic groups in which other deletions and point mutations of the a-globin gene were more common (Rund et al., 2004). In addition, other hemoglobinopathies such as sickle cell anemia and b-thalassemia are rare in Ashkenazim (Oppenheim et al., 1993) while a higher rate might be expected if malarial
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
selective pressure existed in the geographic regions in which they originated. This suggests that founder effect may be the primary reason for the high prevalence of -a3.7 chromosomes in Ashkenazi Jews. In this study we aimed to elucidate the genetic mechanism(s) responsible for the high frequency of a-thal in Ashkenazi Jews, based on genetic analysis of the a-globin gene cluster in this ethnic group including analysis of -a3.7 subtype and haplotypes. We hypothesized that we would be able to determine if a founder effect would explain the frequency, as has been found with other genetic diseases common in this ethnic group (Motulsky, 1995). We also sought, if possible, to clarify the origin of the deletion in this ethnic group by comparing the genetic analysis of Ashkenazim to that of other ethnic groups in our population. 2. Material and methods For over 20 years, our laboratory has been a referral center for DNA based analysis of thalassemia patients from all over Israel. As such we performed globin gene analysis on several hundred individuals found to have microcytic anemia who were not carriers of beta thalassemia trait using hemoglobin electrophoresis or DNA sequencing. This was the referral patient population for this study. The Institutional Helsinki Committee gave its approval for this research. DNA was isolated from peripheral blood leukocytes according to standard procedures (Goossens and Kan, 1981). PCR was performed for the -a3.7 allele as previously described (Oron-Karni et al., 1998). We selected all samples of individuals found to be homozygous for the -a3.7 allele. We reasoned that homozygotes would be a genetically homogeneous group which would facilitate haplotyping. Using PCR and restriction enzyme analysis with ApaI and RsaI according to published protocols (Higgs et al., 1984), we determined the specific 3.7 kb deletion subtype (Fig. 1). We then performed haplotype analysis of the a-globin gene locus based on 9 distinct published polymorphic sites (Miles et al., 2001), 8 of which are present on the -a3.7 chromosome. 3. Results We evaluated 174 chromosomes of 87 -a3.7 homozygotes from several ethnic groups: Ashkenazi Jews: 55 chromosomes, Yemenite Jews: 54, Iraqi Jews: 11, Arabs: 28, Druze 6 and other Sephardi Jews: 14. Ethnicity was self-declared, and only seven individuals declared mixed ethnicity. Using PCR and digestion with ApaI and RsaI, it was determined that 163 of 170 (96%) chromosomes are of the -a3.7I type. An additional 4 alleles were of the -a3.7II type; one Yemenite and 2 Ashkenazi -a3.7I/-a3.7II heterozygotes and one Arab -a3.7II/-a3.7II homozygote. Two samples showed heterozygosity for both ApaI and RsaI restriction, and -a3.7 subtype could not be elucidated, possibly due to an additional crossing over event (Law et al., 2006).
3
Full haplotype restriction pattern was elucidated for 82 samples (94%). In 5 Ashkenazi samples, 1 Yemenite and 1 sample of other origin a full restriction pattern could not be achieved due to technical difficulties. Samples with homozygous haplotypes could be directly identified using restriction sites (Table 2). Two distinct haplotypes could be identified in samples with single restriction site heterozygosity. 73/174 (42%) of chromosomes could thus be identified showing 12 different haplotypes (Ia, Ib, IIa, IIc, IIg, IIh, IIIa, IIIb, IIIe, VIa and 2 newly reported haplotypes). Among Ashkenazim 9 IIIb homozygotes and one IIIb/IIIa compound heterozygote were identified. In total 19/55 (35%) of Ashkenazi chromosomes carried haplotype IIIb. Other than 1 Iraqi homozygote, no other ethnicities exhibited IIIb haplotype. 8 Ia homozygotes and 5 heterozygotes were found in the Yemenite population. In addition 3 IIh homozygotes were found. Among Arabs; 5 Ia homozygotes and one heterozygote were found. 42% of Yemenite chromosomes and 39% of Arab chromosomes carried the Ia haplotype. Based on the high prevalence of Ia in Yemenites and Arabs and IIIb in Ashkenazim we analyzed the remaining samples with multiple restriction site heterozygosity assuming that the prevalent haplotype was present on at least one chromosome (Table 3). Using this analysis 54% of Yemenite and 46% of Arab chromosomes carried the Ia haplotype whereas 67% of Ashkenazi chromosomes carried the IIIb haplotype. In addition to the three Yemenite IIh homozygotes, reanalysis showed 6 probable IIIb/IIh heterozygotes among Ashkenazim and an additional 2 samples that may be heterozygous for IIh. The unavailability of DNA of parents and/or other family members precluded definitive characterization. 4. Discussion In this study we sought to compare alpha globin haplotypes of a group of 87 individuals who were homozygous for the highly prevalent -a3.7 deletion. These individuals were of various ethnicities, primarily Ashkenazi (32%) and Yemenite (31%) Jews. -a3.7 subtyping showed an expected uniform picture with 96% of chromosomes being of the -a3.7I type most commonly found worldwide. Unexpectedly, at the level of the haplotype, a great degree of diversity was found: at least 10 different haplotypes were deduced among Ashkenazim with a large number of chromosomes carrying unknown haplotypes. Interestingly, two thirds of Ashkenazi
Table 3 Common haplotypes and distribution in studied -a3.7 homozygotes following reanalysis based on dominant haplotype. Alleles
Ashkenazim (n ¼ 55)
Yemenites (n ¼ 50)
Arabs (n ¼ 28)
IA IIIB IIH Other
0 37 (67%) 8 (15%) 10 (18%)
27 (54%) 0 (0%) 9 (18%) 14 (28%)
13 (46%) 0 (0%) 0 (0%) 15 (54%)
Table 2 Haplotypes and Distribution in studied -a3.7 homozygotes. Alleles
Ashkenazi Jews (n ¼ 55)
Yemenite Jews (n ¼ 50)
Arab (n ¼ 28)
Iraqi Jews (n ¼ 11)
Druze (n ¼ 6)
Other Jews (n ¼ 24)
Total (n ¼ 174)
IA IIIB IIH Other alleles
0 19 (35%) 0 IIIA e 1
21 (42%) 0 6 (12%) IB e 1 IIC e 3 IIA e 1 IIB e 1 Unknown e 1
11 (39%) 0 e IIC e 1
1 (3%) 2 (18%) e IIIE e 2
0 0 e IIC e 1
1 (4%) 2 (8%) e IIG e 1
33 (19%) 21 (12%) e
IIA e 1 VIA e 1
Unknown e 1
IIA e 2 IIIA e 2
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008
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A. Shaulov et al. / European Journal of Medical Genetics xxx (2016) 1e4
chromosomes carried haplotype IIIb which is common in Melanesia and Papua New Guinea but rarely found in Europe and Saudi Arabia (Flint et al., 1992) (Table 1). Its occurrence in a European ethnic group is not likely to have occurred by migration and more likely occurred by recurrent mutational events at multiple sites. In contrast, only 1.7% of non-Ashkenazi chromosomes carried haplotype IIIb. Nearly half of chromosomes from Arabs and Yemenite Jews carried haplotype Ia. Iraqi Jews and other Sephardi Jewish ethnic groups showed a lower prevalence of Ia while no Ashkenazi samples carried the Ia haplotype. Ia is the most common haplotype globally and the lack of prevalence in Ashkenazim coupled with the high prevalence of the rare haplotype IIIB may provide support for founder effect and genetic drift in Ashkenazim as is the case in other diseases such as Factor XI deficiency, Gaucher, Tay Sachs and others (Motulsky, 1995). Interestingly 3 homozygotes for haplotype IIh were identified in Yemenite Jews, a haplotype previously identified in Aboriginal Australians (Roberts-Thomson et al., 1996). Analysis of results, as described above, based on prevalence of the haplotype common in the ethnic group e Ia in Yemenites and IIIa in Ashkenazim surprisingly identified an additional 3 IIh chromosomes in Yemenites (total prevalence - 18%), and 8 IIh chromosomes in Ashkenazim (prevalence - 15%). That such a rare haplotype may be found in two distinct Jewish ethnic groups which were geographically separated for centuries may well suggest that the IIh is a remnant of a very early founder common to both Ashkenazi and Yemenite Jews. The origin of Ashkenazi Jews has been researched extensively using various genetic tools such as DNA sequencing (Carmi et al., 2014), Y chromosome analysis (Nebel et al., 2005) and mitochondrial DNA (Costa et al., 2013). These have shown a founder effect with Near East genetic imprint as well as variation in genetic markers, as influenced by local populations. Our study of Ashkenazim is unique in that we began by selecting a group of individuals for analysis in whom the genetic evidence pointed to a Middle Eastern origin as evidenced by homozygous -a3.7. We previously showed evidence of high prevalence of the -a3.7 deletion in Ashkenazim which is not prevalent in European populations and yet carries a haplotype distinct from that of other Jewish ethnic groups. The common haplotype, IIh, is distinct from Ia in 4/8 polymorphic sites while IIIa is distinct from IIh in only 2/8. Ia and IIIa differ in 5/8 sites. IIIa may be seen as a result of genetic drift of the common IIh haplotype while Ia is the result of natural selection and the genetic influence of surrounding populations. In summary, haplotype analysis of a homogeneous group of -a3.7 deletion homozygotes showed a dominant and rare IIIa haplotype in addition to a significant population of IIh haplotype chromosomes which may originate from a founder common to other Jewish ethnicities. Haplotype analysis may provide pertinent information about the prevalence of alpha-thalassemia in areas not endemic to malaria. Acknowledgements We thank the Rochlin foundation for their long term support for our research in thalassemia. References
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Carmi, S., Hui, K.Y., Kochav, E., Liu, X., Xue, J., Grady, F., Guha, S., Upadhyay, K., Ben-
Please cite this article in press as: Shaulov, A., et al., Haplotype analysis of a-thalassemia chromosomes reveals heterogeneity and multiple founders in Ashkenazi Jews, European Journal of Medical Genetics (2016), http://dx.doi.org/10.1016/j.ejmg.2016.10.008