Puck, unpublished data) were used to assess array performance. 11– 17 Samples from 59 SCID patients with known SCID mutations and 50 carrier parents 10– 19 (J. 10 Mutations found in 240 consecutive patients with IL2RG mutations enrolled between 19 were used to establish rates of new versus recurrent mutations. Blood samples were subjected to enumeration of lymphocyte subsets, DNA extraction, and transformation with Epstein-Barr Virus (EBV) to create B cell lines, 9 which in turn were used to prepare additional DNA and to measure STAT5 phosphorylation in response to IL-2 stimulation. Some parents were enrolled for carrier determination whose SCID offspring were deceased with no stored tissue. Patients with SCID and their parents, if available, were enrolled with informed consent in IRB-approved protocols to determine genotype and genotype/phenotype correlations. We investigated whether direct detection of known mutations versus resequencing would be appropriate for SCID mutation detection and developed an array-based resequencing approach. Molecular diagnosis of SCID mutations is challenging because a large number of deleterious mutations can be found in each gene, and multiple genes can be mutated to give the same phenotype. Newborn screening for SCID is a desirable public health measure to identify affected infants at a preinfectious stage of their disease, but until this is instituted, physician awareness and ready access to definitive diagnosis are critical so that affected infants can be diagnosed in time for HSCT therapy. Moreover, their early infections may not be distinguishable from those in otherwise healthy infants. 6 However, most SCID infants lack a positive family history and appear normal at birth. Between 5% and 15% of cases are caused by defects in each of IL7R, encoding the IL-7 receptor α chain, and JAK3, encoding an intracellular Janus activating kinase associated with the γc cytoplasmic tail.Įarly diagnosis of SCID, before the onset of severe infections, and prompt treatment in the first months of life bring about the best outcomes.
X-linked IL2RG is mutated in approximately half of all SCID cases and encodes the common γ chain, γc, found in receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These T −B + SCID cases are almost all due to defects in one of three genes that encode lymphocyte receptors and mediators of cytokine signals.
5 All patients with SCID have very low T cell numbers, but about two thirds have normal or even elevated numbers of B cells which, however, fail to produce specific antibodies. To date at least 12 distinct SCID disease genes have been identified. 1– 4 Infants with SCID develop severe infections and do not survive beyond their first years of life unless provided with functional immunity, such as by hematopoietic stem cell transplantation (HSCT) from a healthy donor. Severe combined immunodeficiency (SCID) describes a group of heritable diseases characterized by a profound lack of both T cell and B cell immunity. Future customization of probe sequences and analysis algorithms could increase the number of accurately called nucleotides. 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).Ĭonclusion: Microarray resequencing is a promising technology for severe combined immunodeficiency mutation diagnosis that can detect both known and new mutations. 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. Results: New, unique severe combined immunodeficiency mutations are frequent. We tested males and their mothers with X-linked IL2RG variants and patients and carriers with autosomal variants in IL7R, JAK3, and DCLRE1C. DNA samples were analyzed by array versus standard dideoxy genomic sequencing. 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. A resequencing microarray could facilitate mutation detection, increasing the chance of diagnosing infants early for optimal rescue by hematopoietic stem cell transplantation. 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.