Carrier and prenatal diagnosis of X-linked severe combined immunodeficiency: mutation detection methods and utilization

Abstract

IL2RG, the gene encoding the common gamma chain, gamma c, of the receptor for interleukin-2 and other cytokines, has been identified as the disease gene for severe combined immunodeficiency (SCID) of the X-linked type. Specific mutational diagnosis for X-linked SCID has thus become possible. For many women at risk for carrying an IL2RG mutation, no samples were saved from an affected male relative prior to either death or bone marrow transplantation (BMT). To establish optimal methods for genetic evaluation of such women, we compared mutational screening by single-strand conformational polymorphism, heteroduplex analysis and dideoxy fingerprinting (ddF). Abnormally migrating band patterns were followed up with direct sequencing for identification of specific mutations. The most sensitive method, ddF, detected heterozygous alterations, subsequently confirmed to represent significant mutations, in all of 19 unrelated obligate or suspected carriers studied. Some of these women, as well as others at risk for carrying an X-linked SCID mutation, enrolled in a study of prenatal diagnosis after fetal testing for gender determination. Originally using linkage analysis and, more recently, specific detection of IL2RG mutations, we evaluated pregnancies at risk for X-linked SCID prospectively on a research basis. Of 27 male fetuses tested 14 were predicted to be unaffected and confirmed to have normal immune status at birth. Among pregnancies predicted to be affected, 2 were terminated, while 11 affected males were born at term. Nine of these received neonatal BMT, one had BMT at 3 months of age, and one underwent a successful experimental in utero BMT. In our study cohort accurate prenatal diagnosis assisted decision making and expanded treatment options for families at risk for having infants with a severe, but treatable genetic disorder that presents early in life.

Hematopoietic stem cell transplantation for severe combined immunodeficiency in the neonatal period leads to superior thymic output and improved survival

“…Prenatal diagnosis is recommended for families who are considering terminating an affected pregnancy, to alleviate anxiety, or to allow families time to plan for postnatal treatment of affected infants. 17 Alternatively, all infants could be screened for SCID at birth even when there is no family history; this could be done by routine performance of a white blood cell count and a manual differential count and then flow cytometry if lymphopenia is present. 1,4 Each patient in this analysis was initially evaluated for SCID because one or more family members was affected by the disease.…”

Mutations in genes required for T-cell development:IL7R, CD45, IL2RG, JAK3, RAG1, RAG2, ARTEMIS, and ADA and severe combined immunodeficiency: HuGE review

“…Female carriers of IL2RG mutations can be identified by nonrandom X-chromosome inactivation in lymphoid cells 87 and a variety of other molecular techniques. 86 …”

Identification of single nucleotide polymorphisms (SNPs) and other sequence changes and estimation of nucleotide diversity in coding and flanking regions of the NMDAR1 receptor gene in schizophrenic patients

“…Extensive analyses have demonstrated that ddF and biddF detect virtually 100% of mutations with high specificity. [14][15][16][17] For either ddF or bi-ddF, PCR products were pretreated with 10 U exonuclease I and 2 U shrimp alkaline phosphatase and incubated at 37°C for 15 min followed by a termination step of 80°C for 15 min. A total of 50-500 ng of the treated product was used in conjunction with the Thermosequenase Radiolabled Terminator Cycle Sequencing kit (Amersham, Arlington Heights, IL, USA) according to manufacturer's specifications to generate one of the four standard dideoxy sequencing reaction products.…”