Molecular Analysis of WASP Gene - First Report from India.

Abstract Wiskott Aldrich Syndrome (WAS) is an X-linked recessive disorder associated with severe thrombocytopenia, eczema, bloody diarrhea, profound immunodeficiency and an increased risk of malignancies. More than 200 mutations have been described in the gene encoding the WAS protein (WASP). We report here the molecular basis of WAS in six patients from India. Diagnosis of WAS/X- linked thrombocytopenia (XLT) was based on low platelet count (9000– 1, 25,000/mm3), presence of small platelets (mean: 6.2fl) and detection of WASP by flowcytometry. Patients evaluated in the study could be classified as classical WAS (n=3) and XLT (n=3) depending on the clinical severity of their disease (Zhu et.al 1995). All 12 exons and flanking splice-sites of WAS gene were amplified by polymerase chain reaction (PCR) using primers and PCR conditions adopted with modifications from Kwan et al (PNAS1995;92:4706). Mutation analysis was accomplished by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Abnormal banding pattern was observed only in 4 patients after CSGE analysis. c.995 C>T could not be detected by CSGE but was detected by direct sequencing. The types of molecular defects identified in five patients were as follows: nonsense - 2, deletion - 1 and duplication - 1. Three of these alterations (see table) have been reported previously and are associated with classical WAS. In patients detected to have nonsense mutations, flowcytometric analysis of WASP exhibited high values (table) not consistent with reported western blotting data in these mutations. c.307+10-11dupC was observed in two patients (brothers) with thrombocytopenia, small platelets and mild bleeding. This duplication is not a reported polymorphism. Though this duplication changes the acceptor splice site prediction score from 0.83 to 0.91, its functional significance has not been determined and requires further evaluation. The sixth patient in our study had thrombocytopenia (15,000–45,000/mm3), small platelets (4.8–7.2 fl), mild bleeding, similar family history and normal expression of WASP (86.2%). No mutation has been identified in the coding region of WAS gene in this patient either by CSGE or direct sequencing. Our data shows that the molecular basis for WAS is heterogenous in India. This is the first study evaluating mutations in WAS gene from India. Further mutation screening of patients with WAS will allow the genetic definition, phenotype-genotype corelation, carrier detection and prenatal diagnosis of this condition. Molecular defects observed in WAS patients Patient ID Flow cytometry (%) Mutation *-Reported mutation, ♦-Functional Significance not known PD-22 WASP D1-98.5, WASPB9 - 94.8 c.665C>T, p.Arg211X* PD-16 WASP B9- 6.7 c. 1035-1036 delG, p.Gly314ValfsX120* PD-21 WASP D1-97.2, WASP B9-91.2 c.995 C>T, p. Arg321X* PD-34, PD-35 (siblings) WASP D1-3.6, WASP B9-2; D1-1.8, B9-1 c.307+10-11 dupC♦ PD-28 WASP D1 - 86.2 Mutation not detected