Recent Eastern European ancestry of the F11 gene type I mutation

1. Recent Eastern European ancestry of the F11 gene type I mutation Hava Peretz, Ariella Zivelin, Michal Zucker, Sali Usher, Uri Seligsohn, and Ophira Salomon Clinical Biochemistry Laboratory, Tel Aviv Sourasky Medical Center and Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Israel

2. Coagulation Factor XI (FXI) deficiency is a mild, injury-related autosomal recessive bleeding disorder. It is rare worldwide, yet it is one of the most frequent genetic disorders among Ashkenazi Jews (AJ). The F11 gene is located on chromosome 4q35, it is 23 kb in length and is comprised of 15 exons and 14 introns (A-M). INTRODUCTION

3. Three types of F11 gene point mutations were detected in FXI deficient AJ patients Note the first patient found in 1989to carry the type I mutation Asakai R et al PNAS 86:7667, 1989

4. Mutant allele frequency in 414 unrelated Israeli Jewish patients with severe FXI deficiency The type I mutation is rare compared to the type II and type III mutations *Updated in November 2010

5. Frequency distribution of FXI gene haplotypes observed in Ashkenazi Jews, Iraqi Jews, and Arabs. Ancient Middle Eastern origin of the type II mutation, and a more recent European origin of the type III mutation Scheme showing the common origin of the three major segments of contemporary Jews and explaining the current distribution of the type II and type III mutations. The predicted time when type II and type III mutations occurred in the FXI gene are indicated by horizontal arrows. We speculate that gene flow has been responsible for the transfer of type II mutation from Middle Eastern Jews to Palestinian Arabs after the settlement of Arabs in Israel in the Seventh century Frequency distribution of FXI gene haplotypes observed in Ashkenazi Jews, Iraqi Jews, and Arabs. Note that all type II chromosomes (AJ, non-AJ, Arab) share a common haplotype and type III chromosomes confined to AJ carry another distinct haplotype Peretz H et al Blood 90:2654,1997 Goldstein DB et al Am J Hum Genet 64:1071, 1999

6. OBJECTIVES • Establish the frequency of the type I mutation in the AJ population living in Israel • Test the hypothesis that the type I mutation is a founder mutation • Estimate the geographic/time of origin of the type I mutation

7. METHODS • Subjects • 14 FXI deficient index cases carrying the type I mutation that belong to 13 apparently unrelated Israeli AJ families • 29 related family members • 436 AJ control individuals mostly of Polish Russian and Romanian extraction • Genomic DNA was extracted from peripheral blood cells. • The type I mutation was tested by real time PCR and melting analysis of labeled anchor and sensor primers on a LightCycler machine (Figure 1). • SNPs were analyzed by PCR-RFLP assays or by PCR-direct sequencing. Microsatellite repeats were analyzed by using fluorescent primers followed by size determination with an ABI sequence analyzer. • Eleven families were thoroughly interviewed about the origins of their ancestors.

8. Figure 1. Real-time PCR melting analysis assay used for detection of the type I mutation homozygous wild type homozygous mutant heterozygous carriers

9. Figure 2. Position on chromosome 4 of the genes/loci that were analyzed for polymorphic markers -D4S171 1.3 Mbp -TLR3 -DKFZ -CYP4V2 -F11 4q35 -MTNR1

10. RESULTS • No carriers of the type I mutation were detected in 436 control AJ DNA samples. • Analysis of polymorphic markers within and surrounding the F11 gene, together spanning ~1.3 Mb (Figure 2) revealed a conserved haplotype associated with the type I mutation covering 0.45 Mb (Figure 3, Table 1). • According to the reported history of the interviewed families, the origins of the type I mutation could be traced back to at least 200 years to an Eastern European area surrounding the Carpathian Mountains (Table 2, Figure 4).

11. Ukraine/Galicia(Lvov/Lemberg) Romania(Transylvania) 2nd or 3rd cousins 3 Romania(Maramures) 2 3 FXI 41% FXI 31% 20 20TTA GCCT CW WA C11 9G AG AC T9 8I -G G 20 20CTA AAAC TW WA A11 9G AG AC T8 9II -G G WY KY 4 2 FXI 1% FXI < 1% FXI 40% 19 22TTA ACAC TW WA A11 9G AG AC C9 9I -G A 19 20TTA ACCC CW WA A11 11G GG GC C9 9I IG G 20 20TCA ACAC CW WA A11 11G GG GC C9 8I IIG G D4S171TLR3DKFZCYP4V2 9 F11 MTNRIA Figure 3. Pedigree, results of haplotype analysis and reported origins of family III • The haplotypes associated with the type I mutation are highlighted in red. • The Roman numbers in the haplotype denote the type of the F11 gene mutation • The dotted symbols represent carriers and obligatory carriers of the type I mutation

12. Table 1. Haplotypes associated with the type I mutation in 13 FXI deficient families A,T,G,C stand for the respective polymorphic nucleotides, W for wild type, and the numbers represent the number of dinucleotide repeats. The suggested ancestral haplotype is shown in red

13. Table 2. Reported origins of the studied families In parenthesis are shown the number of generations back from present except for families X and XII where the numbers represent year of arrival to Israel and year of parents’ birth, respectively.

14. What can be learned about the origin of the type I mutation from the family reports? • The best definition of the geographic area that links the places of origin of 11 families would be the Eastern European lands surrounding the Carpathian Mountains. These include the historical regions of Galicia, Transylvania, Bukovina, Moldova and Bessarabia (today Poland, Ukraine, Slovakia, Hungary, Romania, Moldavia). • From the history of family III we can learn that the grand-grand parents of both index cases lived in Transylvania more than 100 years ago, but the origin of the mutation is more ancient and it comes from Lemberg (Lvov)-Galicia (Ukraine). • Family X reported that the ancestors of the index case from the maternal and the paternal side immigrated from Romania to Israel in 1780 and early 1800, respectively, meaning that the mutation was present in Romania more than 200 years ago. • Given the above mentioned information and the history of the last centuries of the region we may speculate that the mutation occurred somewhere in Galicia and spread to Romania that at times included Transylvania, Bukovina, Moldova and Bessarabia.

15. Figure 4. Map showing the probable geographic dispersion of the type I mutation in the last 200 years Approximate area of dispersion of the type I mutation deduced from the reports of 11 families (Table 3)

16. SUMMARY • A common haplotype of polymorphic markers associated with the type I mutation indicates a founder effect. • The low frequency and the limited geographic spread of the type I mutation suggests a relatively recent origin. • Given the family histories and the history of the region we speculate that the mutation originated more than 200 years ago in Galicia and spread to greater Romania. • An attempt will be made to estimate the age of the mutation using mathematical analysis of the molecular data

17. CONCLUSION The population genetics of the F11type II, type III and type I mutations mirror important land-marks in the Jewish history: the foundation of the Jewish people in the Middle East more than 2000 years ago, the consolidation of the AJ in Central Europe around 1000 years ago and a more recent founder event in Eastern Europe, respectively, as was previously observed in other AJ genetic diseases [Risch N, Am J Hum Genet 72:812, 2003].