Lowe syndrome carrier testing by denaturing HPLC. T. Lin, S.F. Suchy, R.L. Nussbaum. Genetics Dis Res Branch, NHGRI/NIH, Bethesda, MD.
   The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked genetic disorder involving the eyes, kidney, and nervous system. The OCRL1 gene encodes ocrl1, a 105 kD phosphatidylinositol (4,5) bisphosphate [PtdIns (4,5)P2] 5-phosphatase that is deficient in OCRL patients. The diagnosis can be confirmed in affected males by enzyme assay, however the biochemical assay cannot be used for carrier detection. While a slit-lamp examination of the ocular lens has been shown to be a highly sensitive (>97%) method for detecting carriers; it requires an experienced ophthalmologist. A more consistent method for carrier detection is essential. Because most families have unique mutations, carrier diagnosis by molecular analysis is limited to families in which the mutation is known or in which linkage is informative. Thus a specific DNA analysis must be tailored for each family. Denaturing HPLC (DHPLC) is a heteroduplexes based, highly sensitive (92-96%) method for detecting unknown mutations. DHPLC is reportedly more sensitive than SSCP, DGGE and PTT and is less expensive than direct sequencing. Among the 73 reported mutations in the OCRL1 gene (23 exons), 93% of the mutations are located in exons 10-23 and 20.5% are in exon 15. We have designed new PCR primers for each exon from 10 to 23. The resulting amplicons are 173 to 807 bp. Our protocol for identification of unknown mutations by DHPLC is to screen exon 15 followed by the remaining exons 10 to 23. The mutated exon will then be sequenced. We have identified two new mutations by this method. One is a single nucleotide insertion (T) between 2498-2499 in exon 22 that causes truncation of the protein and a loss of PtdIns (4,5)P2 5-phosphatase activity. A second mutation, in exon 15, was identified as a 1547G>T missense mutation that causes a loss of enzyme activity.