Diagnostic approaches to apparent homozygosity.

PURPOSE: Sanger sequencing is a mainstay for the identification of gene mutations used in molecular diagnostic laboratories. However, in autosomal recessive disorders, failure of allele amplification can occur for a variety of reasons, leading heterozygous mutations to appear homozygous. We sought to investigate the frequency at which apparently homozygous mutations detected by Sanger sequencing in our laboratory appeared homozygous due to other molecular etiologies.
METHODS: A review of 12,406 cases from 40 different genetic tests that were submitted to the Medical Genetics Laboratories at Baylor College of Medicine for Sanger sequence analysis was performed. The molecular status of apparently homozygous cases was further investigated by testing parents using various methods.
RESULTS: A total of 291 cases of apparent homozygosity were identified, ranging from 0 to 37% of the total per gene. One-third of the apparently homozygous cases were followed up by parental testing. Parental carrier status was confirmed in 88% of the cases. Of the cases in which parental carrier status could not be confirmed, deletions encompassing point mutations, allele dropout due to single-nucleotide polymorphisms at primer sites, and uniparental isodisomy were observed.
CONCLUSION: For individuals with autosomal recessive disorders and apparently homozygous mutations, confirmation by parental testing can rule out other causes of apparent homozygosity, including allele dropout, copy number variations, and uniparental isodisomy.