Purpose: Mutation diagnosis of severe combined immunodeficiency is challenging because of the multiplicity of disease genes and large number of disease-causing mutations, including unique ones that continue to be found. A resequencing microarray could facilitate mutation detection, increasing the chance of diagnosing infants early for optimal rescue by hematopoietic stem cell transplantation.
Methods: After analyzing cumulative mutations, we developed a custom Affymetrix GeneChip microarray including probes representing exons and flanking regions of severe combined immunodeficiency disease genes. DNA samples were analyzed by array versus standard dideoxy genomic sequencing. We tested males and their mothers with X-linked IL2RG variants and patients and carriers with autosomal variants in IL7R, JAK3, and DCLRE1C.
Results: New, unique severe combined immunodeficiency mutations are frequent. Resequencing array call rates of 95-98% exceeded GeneChip product specifications, and all of 47 point mutations in known samples were detected, as were the sites of 12 of 22 disease-causing insertions and deletions. Each gene had particular nucleotides that were often not called correctly and had to be excluded from analysis; exclusion rates ranged from 0.4% (hemizygous IL2RG) to 9.2% (heterozygous JAK3).
Conclusion: Microarray resequencing is a promising technology for severe combined immunodeficiency mutation diagnosis that can detect both known and new mutations. Future customization of probe sequences and analysis algorithms could increase the number of accurately called nucleotides.