2.4. Haplotype Analysis

A haplotype analysis was performed on 190/332 probands for 19 of the most recurrent variants, where one variant was observed in at least four probands with DNA available. Haplotypes were determined by analyzing a total of seven markers, three short tandem repeat (STR) markers (~34.75–36.51 cM, chrX:17741821-19054560 GRCh37) and four single nucleotide polymorphism (SNP) markers (~34.56–36.25 cM, chrX: 17568516-18876226 GRCh37), surrounding the RS1 gene (~1.95 cM, 34.56–36.51 cM, chrX: 17568516-19054560 GRCh37) on the X chromosome (Supplementary Table S3). Highly polymorphic STR markers were evaluated and selected by heterozygosity and proximity to the RS1 gene from the Rutgers Combined Linkage-Physical Map v3 map [26]. Primer sequences were obtained from UCSC genome browser [27] (https://genome.ucsc.edu/, accessed on 26 March 2018). Primer pairs were designed with M13 tails on the forward primers and combined with M13 tagged 5′ 6-fluorescein amidite (FAM), as described previously [28]. A PCR was performed using proband DNA, OneTaq^® Hot Start 2X Master Mix with Standard Buffer (New England Biolabs, Ipswich, MA, USA) and the labeled primers. PCR products were analyzed by capillary electrophoresis using a 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA), marker alleles were called using the GeneMapper ™ software (Applied Biosystems, Waltham, MA, USA), and alleles were determined based on fragment sizes. SNP markers with allele frequencies ranging from ~19% to ~68% were selected from gnomAD v2.1.1 based on the allele frequency in European and Hispanic populations and proximity to the RS1 gene. Primer sequences were designed using Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/, accessed on 18 November 2018) and engineered with M13 tails. PCR and Sanger sequencing were performed following the BigDye Direct Cycle Sequencing protocol (Applied Biosystems, Waltham, MA, USA). Sequencing products were processed by capillary electrophoresis using a SeqStudio Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Sequencing data were analyzed using a Mutation Surveyor^® (SoftGenetics, State College, PA, USA), and letter-based alleles were assigned to the observed nucleotide at the SNP.