©American College of Medical Genetics and Genomics BRIEF REPORT Diagnostic approaches to apparent homozygosity Megan L. Landsverk, PhD1, Ganka V. Douglas, PhD1, Sha Tang, PhD1, Victor W. Zhang, MD, PhD1, Guo-Li Wang, PhD1, Jing Wang, MD1 and Lee-Jun C. Wong, PhD1 Purpose: Sanger sequencing is a mainstay for the identification of apparently homozygous cases were followed up by parental testing. gene mutations used in molecular diagnostic laboratories. However, Parental carrier status was confirmed in 88% of the cases. Of the in autosomal recessive disorders, failure of allele amplification can cases in which parental carrier status could not be confirmed, dele- occur for a variety of reasons, leading heterozygous mutations to tions encompassing point mutations, allele dropout due to single- appear homozygous. We sought to investigate the frequency at which nucleotide polymorphisms at primer sites, and uniparental isodisomy apparently homozygous mutations detected by Sanger sequencing were observed. in our laboratory appeared homozygous due to other molecular etiologies. Conclusion: For individuals with autosomal recessive disorders and apparently homozygous mutations, confirmation by parental testing Methods: A review of 12,406 cases from 40 different genetic tests can rule out other causes of apparent homozygosity, including allele that were submitted to the Medical Genetics Laboratories at Baylor dropout, copy number variations, and uniparental isodisomy. College of Medicine for Sanger sequence analysis was performed. The molecular status of apparently homozygous cases was further investi- Genet Med 2012:14(10):877–882 gated by testing parents using various methods. Results: A total of 291 cases of apparent homozygosity were identi- Key Words: allele dropout; apparent homozygosity; array CGH; fied, ranging from 0 to 37% of the total per gene. One-third of the autosomal recessive; SNP array INTRODUCTION set of primers is used for PCR amplification of regions of inter- The occurrence of true homozygous mutations is not unusual for est. As a result, a single-nucleotide polymorphism (SNP) pres- some autosomal recessive (AR) disorders with common muta- ent within a primer site may disrupt the binding of that primer, tions, such as medium-chain acyl-CoA dehydrogenase defi- and allele dropout could unknowingly occur giving the appear- ciency, cystic fibrosis (CF), and hereditary hemochromatosis.1–4 ance of homozygosity. If a heterozygous deletion is located Homozygous mutations are also more likely to be detected in the same region of amplification, only one chromosome in consanguineous families or small populations. However, is amplified, which would also result in the region of interest numerous publications have reported mutations that appeared appearing homozygous. In uniparental isodisomy (UPD), two to be homozygous yet had another underlying genetic cause.5–8 identical copies of the same chromosome are inherited from Therefore, whether observed homozygosity is a result of an indi- one parent. Therefore, although UPD causes true homozygosity vidual carrying the same mutation on both chromosomes, or a of a point mutation, the molecular mechanism is different than product of two different genetic events, can be investigated by if a proband inherited the same mutation from both parents, testing the parents of the affected individual. and the two mechanisms have very different recurrence risks Targeted analyses for specific common mutations such as (almost 0% in the case of UPD but 25% in the case of identity the c.985A>G (p.K329E) mutation in medium-chain acyl-CoA by descent). dehydrogenase deficiency and the cystic fibrosis deltaF508 Apparent homozygosity is a phenomenon observed in every mutation can be performed using a variety of molecular tech- molecular diagnostic laboratory. Nevertheless, to our knowl- niques including but not limited to restriction fragment length edge, its frequency and the molecular etiology behind it have polymorphism, allele-specific oligonucleotide, pyrosequencing, never been systematically studied. To determine the rate at and amplification refractory mutation system. However, if there which apparently homozygous mutations detected in our labo- are no frequently appearing mutations in the genes of interest, ratory were truly homozygous, we reviewed 291 cases of appar- Sanger sequence analysis remains the standard methodology ent homozygosity from 40 different AR genetic diseases tested for DNA sequence analyses in most molecular diagnostic labo- in our laboratory. All of the cases reviewed were analyzed by ratories. Regardless, all of these techniques rely on PCR-based PCR amplification and Sanger sequencing. Genes in which tar- amplification of a particular genomic region. In general, a single geted testing for specific mutations, such as the medium-chain 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA. Correspondence: Lee-Jun C. Wong ([email protected]) Submitted 30 November 2011; accepted 23 April 2012; advance online publication 17 May 2012. doi:10.1038/gim.2012.58 GENETICS in MEDICINE | Volume 14 | Number 10 | October 2012 877 BRIEF REPORT LANDSVERK et al | Diagnostic approaches to apparent homozygosity acyl-CoA dehydrogenase deficiency c.985A>G (p.K329E), was genome was ~400–500 kb per probe. Samples were processed as performed were not included in this study. previously described using 1 µg of patient DNA.9 Although further testing was recommended by the laboratory in every case, only about one-third of the cases were followed Detection of UPD by SNP array up by the referring clinician through testing of parental samples. The Human610-Quad SNP array, which covers 550,000 SNPs Of those cases, we found that nearly 12% did not inherit the plus an additional 60,000 genetic markers per sample, was mutation from each parent. Our results underscore the impor- used as previously described10 per the manufacturer’s protocol tance of further confirmation and investigation of apparently (Illumina, San Diego, IL). Genotyping data was analyzed for homozygous mutations detected by Sanger sequencing. copy-neutral absence of heterozygosity with GenomeStudio software using the CNV partition 2.3.4 algorithm (Illumina). MATERIALS AND METHODS Patients and DNA RESULTS Tissue or blood samples from patients with clinical diagno- The results of 12,406 reports from testing 40 different AR genes ses of AR diseases were submitted to the Medical Genetics related to mitochondrial or metabolic diseases were reviewed Laboratories at Baylor College of Medicine for molecular eval- (Table 1). A total of 291 cases (~2%) with apparently homozy- uation between January 2007 and December 2010. A major- gous point mutations or small insertions/deletions detected by ity of the samples analyzed were blood. Of the 291 samples of Sanger sequencing were identified. The percentage of apparently apparent homozygosity other sample types included muscle homozygous cases per gene ranged from 0–37%, with a majority (4), skin fibroblast cultures (2), and liver (2). Four samples were of genes having less than 10%. Of note, the highest percentage of received as DNA extracted from blood at the referring institu- apparently homozygous cases was in the ARG1 gene (13/35), in tion. Total genomic DNA was extracted from peripheral blood which 10 different mutations, distributed throughout the gene, leukocytes or other tissues using a commercially available DNA were observed. Of these 13 individuals, 10 were Hispanic and isolation kit (Gentra Systems, Minneapolis, MN) according to 3 were from the Middle East with family histories suggesting the manufacturer’s protocols. A 260/280 absorbance measure- different degrees of consanguinity. In fact, a majority of cases ment between 1.7 and 2, a 260/230 absorbance measurement for the four genes that had the highest percentage of apparent greater than 1.5, and a concentration of a minimum of 50 ng/µl homozygosity (ARG1, FAH, MMACHC, and CPS1) were from were required for further PCR analysis of DNA samples. A sec- populations in which consanguinity is not uncommon. ond extraction was performed if samples were not of sufficient For those genes in which more than one case of apparent quality, and also to confirm positive findings. For those samples homozygosity was detected, only three had less than 50% muta- received as extracted DNA, the original sample was run a sec- tion diversity: POLG (7/25), ALDOB (3/10), and HADHA (1/7) ond time. This study was approved by the institutional review (Table 1). The most frequently observed mutations in ALDOB board of Baylor College of Medicine. and HADHA are the common mutations c.448G>C (p.A150P) (8 of 10 patients) and c.128G>C (p.E510Q) (seven of seven Sequence analysis patients), respectively. Sequence analysis of the appropriate gene(s) was carried out Every report with a result of an apparently homozygous using gene-specific primers to amplify coding exons and at least mutation that was issued from our laboratory suggested paren- 50 intronic nucleotides flanking each exon, and sequencing was tal samples be submitted for carrier confirmation. However, we performed as previously described.8 DNA concentrations were received both parental samples in only ~26% (75/291) of the consistent throughout all PCR reactions at ~50 ng. For those apparently homozygous cases. In an additional eight cases, the samples in which alternate primers were designed, primer sites mothers of the probands were tested and confirmed to be car- outside