Exonic duplication of the OTC gene by a complex rearrangement that likely occurred via a replication-based mechanism: a case report

Sanger sequencing was performed to screen for genetic variations at the nucleotide level throughout all coding exons of the OTC gene (Additional file 1). We used UCSC genome browser (http://​genome-asia.​ucsc.​edu/​) as human genome assembly. To screen for exonic deletions or duplications, multiplex ligation-dependent probe amplification (MLPA) was performed using the SALSA P079-A3 OTC MLPA kit (MRC Holland, Amsterdam, The Netherlands), in accordance with the manufacturer’s recommendations. MLPA products were separated by capillary electrophoresis on an ABI3730 genetic analyzer and then processed using GeneMapper software. The peak heights of the samples were compared with control probes and the ratios of these peaks were calculated for all exons. If the dosage quotient was 1.0, the results were considered normal. Thresholds for deletions and duplications were set at 0.5 and 1.5, respectively.

To demarcate the duplicated region, quantitative real-time PCR was conducted on blood DNA from the patient and a male control subject using the Applied Biosystems 7300 real time PCR system (Thermo Fisher Scientific). Several primer pairs were designed for OTC (upstream of exon 1 and intron 6) and RPP30 that was used as an autosomal single copy gene reference to generate amplicons suitable for real-time PCR (Fig. 1b, Additional file 1). The PCR reaction was performed in a 15 μL reaction system, containing 2 μL of template DNA (5 ng/μL), 0.6 μL of each primer set (10 μmol/L), 0.3 μL ROX Reference Dye, 4 μL distilled water, and 7.5 μL of 2xTB Green Premix Ex TaqII (Tli RNaseH Plus, TaKaRa). Two parallel PCR reactions were prepared for each sample. The amplification cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C 5 s and 60 °C for 1 min. Data evaluation was carried out using the 7300 system SDS software and Microsoft Excel. The threshold cycle number (Ct) was determined for all PCR reactions and the same threshold and baseline were set for all samples. The starting copy number of the samples was determined using theΔΔCt-Method. ΔΔCt method was a modification of the method described in Livak et al. for quantifying mRNA [8]. ΔCt represents the mean Ct value of each sample and was calculated for OTC and RPP30. The starting copy number of the unknown samples was determined relative to the known copy number of the control sample using the following formula:

ΔΔCt = [ΔCt OTC(patient)-ΔCt RPP30(patient)] - [ΔCt OTC(female)-ΔCt RPP30(female)]. The relative gene copy number was calculated by the expression2^-Δ(ΔCt). The starting copy number of male control was also determined as a reference value.

Inverse PCR were performed using restriction enzyme TaqI (TaKaRa, Shiga, Japan) to isolate the unknown sequences adjacent to the duplicated region of the OTC gene in the study patient. ApE – A plasmid Editor software was used to identify the recognition sites for the restriction enzyme. The restriction enzyme was chosen based on the following criteria: (1) no cutting of the expected breakpoint area; and (2) endonuclease activity would be unaffected by CpG methylation of the target sequence. A 100 ng aliquot of genomic DNA from both our patient and a control female was digested with the selected restriction enzyme in a total volume of 30 μl at 65 °C for 90 min. The reaction was inactivated using the QiaQuick PCR Purification Kit. A 20 μL sample of digested DNA was then mixed with 23 μL of DW, 5 μL of 10 × T4 ligase buffer (TaKaRa, Shiga, Japan) and 2 μL of T4 DNA ligase to make a final volume of 50 μL. Ligation reactions were incubated at 16 °C for 16 h. For subsequent PCR, 1 μL of digested and re-ligated DNA template was used in a total reaction volume of 25 μL with Tks Gflex DNA Polymerase (TaKaRa, Shiga, Japan). Primers were designed to avoid repetitive sequences (Additional file 1). The PCR conditions were as follows: 30 cycles of 10 s at 98 °C, 15 s at 60 °C, and 1 min at 68 °C. Amplified products were analyzed by gel electrophoresis and were purified following nested PCR (Additional file 1). The purified PCR products were sequenced via the standard Sanger method.

PCR was performed using Tks Gflex (TaKaRa, Shiga, Japan) to confirm the other side of the breakpoint sequence. Primer R which was previously designed for real-time PCR analysis of OTC upstream of exon 1 (i.e. OTC intron 1) was used as primer F in this reaction (Additional file 1). The PCR conditions and Sanger methodology were similar to those described above.

MLPA revealed the duplication of exons 1–6 of the OTC gene in our current study patient (Fig. 1a). We determined the range of the duplication using quantitative real-time PCR (Fig. 1b). We designed four qPCR experiments (U1-U4) between the promoter and enhancer regions to identify the upstream breakpoint. Likewise, we designed four qPCR assays (D1-D4) within intron 6 to identify the downstream breakpoint. In contrast to the male or female controls that showed ΔΔCt ratios of 0.5 or 1.0, respectively, the patient’s samples showed aΔΔCt ratio > 1.5 in some of these qPCR assays, suggesting that these regions were duplicated in this patient (Fig. 1c). The results indicated that the putative upstream breakpoints were located between PCR U3 and U4, and that the downstream breakpoints were between PCR D2 and D3.

We next performed inverse PCR to analyze the genomic structure of the duplicated region. TaqI-digested DNA was used as a template to produce a 3.5 kb PCR product when amplified with inversely oriented intron 6 primers (Fig. 2a, c). However, an additional small PCR product was detected by agarose gel electrophoresis in the patient sample (Fig. 2a). The amplified products were sequenced after nested PCR (Fig. 2a). As expected, the breakpoint was located within intron 6 (Fig. 2b, c). Unexpectedly however, this breakpoint was found to be connected with intron 1 of the OTC gene in the reverse orientation. The breakpoint junction contained 2 nucleotides of microhomology at the fusion junction (Fig. 2b).

The other side breakpoint was analyzed using standard PCR with primers for the upstream breakpoint region and the breakpoint region in intron 1. The primer pair amplified only products from the patient’s DNA (Fig. 3a). By Sanger sequencing, the upstream region of the OTC gene was found to make an inverted connection with 1 (Fig. 3b, c). This breakpoint junction contained an additional 4 nucleotides (ACTA) of unknown origin (Fig. 3b). The positions of the two breakpoints in intron 1 were found to be chrX: 38365292 and chrX: 38366694, which were 1402 bp apart (Fig. 3c). We performed the same PCR amplification of both junctions in the patient’s parents but detected no products, suggesting that this complex rearrangement arose de novo. The patient’s duplicated region included a common single nucleotide variant (rs752750694, NM_000531.5:c.-844C > T). The patient’s father carries an A whereas the mother carries a G/G at this site (Fig. 3d). The patient was found to be an A/G heterozygote, but the peak of the A nucleotide was two-fold greater than the G-peak, suggesting that the patient carries two copies of A. These data suggest that the de novo duplication was of paternal origin.