Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized kit and quantified by agarose gel electrophoresis.  If gene sequence analysis is requested, all the analyzed regions of the gene are amplified through polymerase chain reaction (PCR) and the exact nature of the gene’s sequence variation(s) can be identified through double-stranded sequencing from sense and anti-sense directions.  If specific mutation analysis is requested, only specific region(s) of DNA is (are) amplified by PCR and sequenced. ARX exons 1-5 plus at least 20 bases into the 5’ and 3’ ends of all the introns are analyzed.  The following sites are used to search for previously described ARX mutations and polymorphisms: Human Gene Mutation Database (HGMD) and online search engines (e.g., PubMed). 

Maternal Cell Contamination (MCC) Assay Information (applies only to prenatal samples tested for MCC): Six different STRs at FGA, TPOX, D8S1179, VWA, D18S51, and CSF1PO loci and up to 10 additional STR loci can be used for MCC study of maternal and fetal DNA using polymerase chain reaction and fragment analysis by capillary electrophoresis. Unresolved cases needing even further study may be forwarded to an outside reference laboratory.  Validation studies in our laboratory have shown that MCC at levels of approximately 5% and higher can be detected by our MCC assay.  Separate studies with our diagnostic sequencing tests have shown that the test and interpretation are not affected until MCC reaches greater than 10%.  Therefore, a negative MCC result is expected to rule out all cases of MCC that may have interfered with our molecular diagnostic result.