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Technical Briefs |
1 Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan
2 Department of Pediatrics, Hospital affiliated with Kanagawa Prefecture School of Nursing and Midwifery, Yokohama, Japan
aaddress correspondence to this author at: Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan; fax 81-53-435-2794, e-mail mmaekawa{at}hama-med.ac.jp
Genetic variants of human butyrylcholinesterase (EC 3.1.1.8; serum cholinesterase; pseudocholinesterase; BCHE) are reported to be associated with prolonged apnea in patients taking the muscle relaxant drug succinylcholine (1) and with low serum BCHE activity (2). The gene encoding BCHE is at least 73 kb long and contains one noncoding and three coding exons (3). Genetic variants of BCHE have been reported (2)(4)(5)(6)(7)(8). We analyzed BCHE mutations in the Japanese population (2)(4)(5)(6). The BCHE mutations found in study populations in the United States have been quite different from those found in Japanese study populations (2)(7). An atypical variant of BCHE has been detected in Caucasians but not in Japanese populations, but fluoride-resistant genes have been reported in Japanese populations as well as Caucasian populations (5)(6).
We recently detected an abnormal genotype in members of a family with low serum BCHE activity. This family carried an insertion mutation in the BCHE gene. Although PCR techniques are now used routinely in genetic testing, there are still possible sources of errors that researchers must be aware of and consider when designing assays. In this report, we present an important example of a pitfall in mutation detection.
Routine laboratory examination identified very low serum BCHE activity (42 U/L) in a 1-year-old boy (reference interval, 4250–7250 U/L). Because secondary hypocholinesterasemia attributable to hepatic dysfunction or organophosphorus poisoning was ruled out on the basis of other biochemical data and clinical symptoms, the child was thought to be homozygous for a silent BCHE gene. The serum BCHE activities in his mother and father were 3009 and 3767 U/L, respectively. Serum BCHE activity was measured spectrophotometrically with butyrylthiocholine iodide (EIKEN CHEMICAL) as a substrate at 37 °C on a JCA-BM2250 automated biochemistry analyzer (JEOL). Genetic analysis was performed after approval from the Institutional Review Board of Hamamatsu University School of Medicine, and informed consent was obtained from all family members.
Genomic DNA was extracted from EDTA-treated venous blood as described by Kunkel et al. (9). Coding exons of the BCHE gene were amplified as nine independent fragments by PCR, and each amplified product was analyzed by single-stranded DNA conformation polymorphism analysis (2)(4) and denaturing HPLC (WAVE System; Transgenomic). PCR products with variant migration patterns were sequenced directly with a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and a PRISM 310 Genetic Analyzer (Applied Biosystems). Bands with different mobilities were excised from the gel and cloned into pDRIVE (Qiagen) for sequencing. The mutation was confirmed by PCR-restriction fragment length polymorphism analysis.
We detected a G-to-C missense mutation at codon 365 (G365R) in exon 2 of BCHE. The proband was homozygous for this mutation, his father was heterozygous, and his mother was homozygous wild type. Representative DHPLC and sequencing results are shown in panels A and B of supplemental Fig. 1, which appears in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue12/. We confirmed our results with PCR-restriction fragment length polymorphism analysis using TaqI (Fig. 2 in the online Data Supplement). If our results are correct, the pedigree and genotype segregations are not easily understood. Hemizygosity of the region containing the G365R site resulting from a large deletion, an inversion, uniparental disomy (genomic imprinting), or a de novo mutation may explain the pedigree. We first tried long PCR to address the possibility of a large deletion, but the result did not provide a clear explanation for the pedigree. We did, however, notice a faint band that migrated more slowly than the target PCR product for exon 2. We therefore changed the extension time of the amplification conditions. The results obtained by electrophoresis on an agarose gel of PCR products from reactions performed with different extension time is shown in Fig. 1
. When longer extension times were used, PCR product was longer. We suspected an insertion mutation and therefore excised the longer fragment from the gel and subcloned and sequenced it. We identified an abnormal sequence inserted in exon 2 as an Alu sequence and a direct repeat (300 + 15 bp). This inserted sequence may have caused premature termination of transcription (Fig. 3 in the online Data Supplement). Both the proband and his mother were heterozygous for this insertion. We therefore concluded that the proband was a compound heterozygote for the G365R missense mutation and the Alu insertion mutation.
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Alu sequences are short interspersed elements that are distributed widely throughout the human genome (10). Alu sequences can be divided into subfamilies of related elements on the basis of diagnostic mutations that are shared by subfamily members. The family described here is the second reported in which insertion of an Alu has caused BCHE deficiency; the first was described by Muratani et al. (8). Although the Alu sequence in the present study was classified as AluYb8 because of similarities with the AluYb8 reported by Muratani et al. (8), it also differed at several nucleotides.
In the present study, if genetic analysis had been performed only on the proband, he would have been misdiagnosed as homozygous for the G365R mutation. Analysis of the family revealed a discrepancy in genotype segregation, which directed us to additional analyses and allowed us to reach the proper conclusion. Shorter PCR programs are convenient for mutation detection, but fragments that are longer than expected may not be amplified and would therefore be missed. We conclude from our present experience that PCR with short extension times may be a source of pitfalls in mutation detection.
References
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