Detection of Carrier Status and Mutations in Family Members of a Child with Complete Deletion of the DAX1 Gene using Multiplex Ligation-Dependent Probe Amplification

Abstract

To the Editor:We have previously reported a case of adrenal crisis [1]. Real-time SYBR Green DNA PCR and gel electrophoresis showed lack of amplification of exons of NRB01/DAX1 (dosage-sensitive sex reversal) gene on Xp21 locus, suggesting a complete deletion. We demonstrated that molecular techniques may prove useful in making a diagnosis and for genetic counselling in adrenal hypoplasia congenital (AHC); information in this letter is supplementary to the article already published [1]. AHC is a rare congenital adrenal disorder where there is primary adrenal failure in early life and hypogonadotropic hypogonadism during adolescence. X-linked AHC is caused by deletions/mutations in DAX1 gene AHC critical region on X chromosome. We performed genetic analysis using Multiplex Ligation-Dependent Probe Amplification (MLPA) on family members of proband, so as to further understand genetic factors involved in inheritance and presentation of this child with AHC, and to detect affected or carrier status in relatives. MLPA, an extension of PCR, allows highly multiplexed detection of relative copy number variations of target sequences in a fast, reliable, and cost-effective manner [2]. Genetic studies in families of patients with affected members have been previously reported [3]. Six family members were tested by MLPA: child’s parents, sister, maternal aunt and her children (Fig. 1). MLPA analysis was performed using SALSA MLPA P185 Intersex probemix (MRC-Holland, Netherlands). Each probe generates an amplification product of unique length that can be detected by capillary electrophoresis. Comparison of relative sizes of the fluorescent peaks from the target probes with reference probes coamplified during the reaction, using analysis software, allows quantification of relative abundance of target regions. N. D. Phadke :K. A. Khatod GenePath Dx (Causeway Healthcare Pvt Ltd), J.M. Road, Shivajinagar, Pune, India