We included the full complement of 829 known protein-coding genes on the X chromosome in the Vega database of manually curated genes in the design. A number of genes on the X chromosome are present in multiple, highly similar copies. Where it was impossible to place PCR amplimers such that only a single copy was amplified, the gene was omitted from the screen. DNA from a single affected individual in each family was PCR-amplified to produce products of approximately 500 bp which were sequenced in both directions on ABI 3730 DNA analysers as previously described34. Sequence variants were called automatically using in-house analysis software, autoCSA35, and also by alignment of base-called data to the X-chromosome reference sequence. We verified all identified mutations by manual inspection of the trace files. All potential truncating variants were further investigated by resequencing of the appropriate sample from a newly amplified PCR product. Because of the large number of missense and synonymous base substitutions it was not practical to confirm these similarly. Therefore, to assess the overall reproducibility of base substitutions, we resequenced 60 and showed that 56 (93%) were confirmed. All missense and synonymous substitutions were therefore included in the analyses.
