Genotyper software version 3

Genomic DNA was extracted from peripheral blood leukocytes using Isolate II Genomic DNA kit from Bioline. Haplotypes were constructed using the following microsatellite markers: D12S1648, D12S2080, D12S2194, D12S2514, D12S2516, D12S2518, D12S2519, D12S2520, D12S1048 and D12S1301, covering an interval of 11.27Mb. The genotyping of microsatellite markers was performed using fluorescently labeled primers, and the products were pooled and analyzed on a 310 Genetic Analyzer using Genotyper Software version 3.7 (Applied Biosystems). The CEPH DNA sample 1331–01 was used as external standard to control for consistency between runs. We used Phase v2.1.1 software [18 (link)] to infer the haplotypic phase for all subjects. The age of the most recent common ancestor of the 36 Arab-speaking and the 15 Berber-speaking G2019S carriers was estimated separately using a likelihood-based method implemented in the ESTIAGE program [19 (link)]. We used allele frequencies obtained from the control individuals and a stepwise mutation model with a mutation rate of 10⁻⁴ at each marker per generation was assumed [20 (link)].

We confirmed the MBD array data using bisulfite direct sequencing. DNA samples (1 μg) were bisulfite-converted using an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s instructions. The bisulfite-converted DNA was then amplified by PCR with primers for discrimination of the methylated and unmethylated CpG sites. The sequences of PCR primers are presented in Table 1. PCR reaction solutions contained 10 ng genomic DNA, 10 pM primers, 0.25 mM dNTPs, 1.5 mM MgCl₂, 1 X buffer, and 0.25 U Taq polymerase per 20 μL of total reaction volume. PCR conditions included predenaturation at 95°C for 10 min, 35 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 40 sec, and final extension at 72°C for 10 min. After PCR amplification, PCR products were purified using a PCR purification kit (Bioneer, Daejeon, Korea) and sequenced using a PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster city, USA) according to manufacturer’s instructions. Sequencing products were analyzed using a PRISM 3100 Genetic Analyzer (Applied Biosystems), and electropherogram traces were interpreted using Genescan software version 3.7 (Applied Biosystems). Corresponding genotypes were assigned using Genotyper software version 3.7 (Applied Biosystems).