No Association Between CEL-HYB Hybrid Allele and Chronic Pancreatitis in Asian Populations

Abstract

A hybrid allele between the carboxyl ester lipase gene (CEL) and its pseudogene, CELP (called CEL–HYB), generated by non-allelic homologous recombination between CEL intron 10 and CELP intron 10′, was found to increase susceptibility to chronic pancreatitis in a case–control study of patients of European ancestry. We attempted to replicate this finding in 3 independent cohorts from China, Japan, and India, but failed to detect the CEL–HYB allele in any of these populations. The CEL–HYB allele might therefore be an ethnic-specific risk factor for chronic pancreatitis. An alternative hybrid allele (CEL–HYB2) was identified in all 3 Asian populations (1.7% combined carrier frequency), but was not associated with chronic pancreatitis.

The etiology of chronic pancreatitis (CP) is complex and involves a subtle interplay between genetic and environmental factors. Most of the hitherto reported genes/loci affecting disease susceptibility encode proteins of the protease-antiprotease system of the pancreatic acinar cells.^1–7 A hybrid allele (CEL–HYB), involving the CEL gene (encoding pancreatic carboxyl ester lipase⁸) and its tandemly linked pseudogene (CELP), was recently reported to be significantly overrepresented in CP cases as compared with controls, firstly in a discovery cohort of patients with familial CP and then in three replication cohorts of patients with idiopathic CP.⁹ The CEL–HYB allele resulted from non-allelic homologous recombination (NAHR) occurring between CEL intron 10 and CELP intron 10′; replacement of the eleventh and last exon of CEL by CELP exon 11′ would yield a premature stop codon within the third “pseudo” 33-bp variable number tandem repeat (VNTR)⁹ (Figure 1A). The mutant enzyme is more stable than its wild-type (WT) counterpart and induced autophagy in cellular models,⁹ suggesting a novel pathogenic mechanism. However, since the patients analyzed in the original study⁹ were solely of European ancestry, replication in independent populations of different ethnicity was warranted.¹⁰

Figure 1.

(A) Key differences between CEL–HYB1 (originally termed CEL–HYB⁹), CEL–HYB2 and the wild-type CEL gene in terms of the defining exon 10, intron 10 and exon 11 sequences. The gene structure of CEL (in black) and that of the replacement CELP sequences (in green) within CEL–HYB1 and CEL–HYB2 were described in accordance with GenBank accession number AF072711.1. In CEL, the VNTRs within exon 11 are indicated in purple; nucleotide positions of the exon 10 boundaries and the beginning of exon 11 are numbered by reference to the A of the translational initiation codon ATG as c.1; the translational termination codon is denoted by a vertical blue bar, with the last coding nucleotide and the amino acid position of the translational termination codon being numbered above and below the bar. In CEL–HYB1, CEL–HYB2a and CEL–HYB2b, the presumed premature stop codons are indicated in a similar manner. (B) A representative gel showing the RT-PCR analyses of HEK293T cells transfected with expression constructs carrying the full-length CEL–HYB1 and CEL–HYB2a genomic sequences. Sanger sequencing of the approximately 2.2-kb CEL–HYB1 and CEL–HYB2a products revealed that all introns were spliced correctly. CV, control vector. (C) Relative mRNA expression levels of CEL–HYB2a versus CEL–HYB1 in vitro as determined by quantitative RT-PCR analyses. CV, control vector. (D) Relative mRNA expression levels of CEL–HYB2a in transfected cells with (grey) and without (black) cycloheximide treatment as determined by quantitative RT-PCR analyses. **, P < 0.01.

The detection of the CEL–HYB allele is dependent upon a long-range duplex PCR assay.⁹ Using French CEL–HYB positive samples, we found that the originally described PCR assay⁹ yielded better results under slightly modified conditions (see Supplementary Material and Supplementary Figure 1). Consequently, these modified conditions were employed to screen for the CEL–HYB allele in three Asian populations. We first analyzed a cohort of Han Chinese patients with idiopathic CP. The previously described CEL–HYB-specific 3.2-kb band was detected in 2.4% (19/799) of patients and 1.9% (20/1028) of controls (P = 0.64; Table 1). Sequencing of the 3.2-kb band-containing PCR products however revealed that none of these Chinese CEL–HYB alleles corresponded to that expected; instead, they invariably resulted from an NAHR event occurring within a 239-bp sequence tract affecting the intron 9/exon 10 boundary of CEL and the intron 9′/exon 10′ boundary of CELP (Supplementary Figure 2). Hereafter, we term the previously reported disease-associated CEL–HYB allele⁹ as CEL–HYB1 so as to distinguish it from this newly described allele, CEL–HYB2.

Table 1.

Carrier frequencies of the CEL–HYB2 variant in Chinese, Japanese and Indian subjects with idiopathic chronic pancreatitis^*

Populations	Case		Control		P valuea
	+/n	%	+/n	%
Chinese	19/799	2.4	20 (1)/1028	1.9	0.64
Japanese	4b/248	1.6	7 (2)/403	1.7	0.85
Indian	0/280	0	1 (1)/225	0.4	0.91
Combined	23/1327	1.7	28 (4)/1656	1.7	0.96

^*Number of subjects carrying the less frequent CEL–HYB2 subtype, CEL–HYB2b, is indicated in parentheses wherever applicable.

^aTwo-tailed Fisher’s exact test.

^bOne patient is a homozygote.

Closer inspection of the 39 Chinese CEL–HYB2 alleles revealed that they could be divided into two subtypes by reference to three single nucleotide polymorphisms (SNPs) present within the substituting CELP sequence (Supplementary Figure 2); 38 harbored the G allele of rs10901232A/G, the T allele of rs10901233C/T and the G allele of rs671412A/G (termed CEL–HYB2a), the sole exception harboring the alternative alleles of these three SNPs (termed CEL–HYB2b). We further genotyped two idiopathic CP cohorts from Japan and India. Results from the Japanese cohort were remarkably similar to those from the Chinese cohort whereas in the Indian cohort, only one control carried a CEL–HYB allele, and this corresponded to CEL–HYB2b (Table 1). Note that a variant similar to CEL–HYB2 was mentioned in passing by Fjeld et al.⁹ to be present in one European case and four controls, although no sequence details were presented. Sequencing of two corresponding French CEL–HYB positive samples revealed their identity to be CEL–HYB2b. In short, the PCR assay employed efficiently detects all three CEL hybrid alleles, the differentiation of which relies upon the sequencing of the respective PCR products.

Both CEL–HYB2a and CEL–HYB2b variants harbor premature stop codons within their chimeric exons 10 (Figure 1A); their corresponding mRNAs could therefore be subject to significant degradation by nonsense-mediated mRNA decay (NMD). However, this might not be the case for CEL–HYB1 because mRNAs that harbor a stop codon in the final exon usually escape degradation by NMD.¹¹ To explore this possibility, we first sought to compare the mRNA expression levels of CEL–HYB2a versus CEL–HYB1 in vitro (Supplementary Material and Supplementary Tables 1 and 2). Briefly, we PCR amplified the full-length genomic CEL–HYB2a and CEL–HYB1 sequences from their corresponding carriers and cloned the resulting PCR products into the pcDNA3.1/V5-His-TOPO vector. Reverse transcription-PCR (RT-PCR) analyses of mRNAs from subsequently transfected HEK293T cells indicated lower CEL–HYB2a mRNA expression as compared to CEL–HYB1 (Figure 1B). Further quantitative RT-PCR analyses demonstrated that the mRNA expression of CEL–HYB2a accounted for only 60% of that of CEL–HYB1 (Figure 1C). Then, we tested whether the mRNA expression level of CEL–HYB2a could be increased by treatment of the transfected cells with cycloheximide, a known NMD inhibitor¹² and found this to be the case (Figure 1D). Hence, we conclude that mRNA expression from the CEL–HYB2a allele is significantly reduced by NMD. This conclusion probably also applies to CEL–HYB2b owing to its high sequence similarity with CEL–HYB2a (Figure 1A).

In summary, contrary to our expectation, we failed to identify the CEL–HYB1 allele in any of the three Asian cohorts. Based on the allele frequency of CEL–HYB1 in healthy German and French populations (0.4%),⁹ the power to detect at least one CEL–HYB1 carrier among the cases is estimated to be >86%, even for the smallest cohort. Given that most rare variants (defined as a minor allele frequency of <0.5%) are population-specific,¹³ CEL–HYB1 may be an ethnicity-specific disease risk factor, although this remains to be confirmed by replication in an independent cohort of European ancestry (e.g., the North American Pancreatitis Study 2¹⁴). The other unexpected finding was the identification of an alternative CEL–HYB2 allele in all three Asian populations. The significant degradation of CEL–HYB2a mRNA by NMD and the observation that pancreatic exocrine function has been found to be normal in Cel-knockout mice¹⁵ are consistent with the apparent lack of any association between CEL–HYB2a and CP.

Supplementary Material

1

Supplementary Figure 1. Comparison of the originally reported PCR assay (A) and the currently used PCR assay (B) for detecting the CEL–HYB1 (as well as the CEL–HYB2b) allele (3.2 kb) in French heterozygotes. WT, wild-type (4.2 kb). NC, negative control.

Supplementary Figure 2. Alignment of reference CEL and CELP sequences in the context of exon 10 (in bold) and partial flanking sequences. The crossover region for the CEL–HYB deletion allele⁹ (termed here CEL–HYB1) is doubly underlined whereas that for CEL–HYB2 is singly underlined. The discriminant positions between the two paralogous sequences are indicated by numbered upwardly pointing arrows. The CEL–HYB2a variant harbors the G allele of rs10901232A/G, the T allele of rs10901233C/T and the G allele of rs671412A/G whereas the CEL–HYB2b variant harbors the alternative alleles of these three SNPs (the three affected sites are indicated by circles). The premature translation termination codon in the chimeric exon 10 of the CEL–HYB2b variant is indicated by a box. The two primers used for sequencing the breakpoint junctions of the CEL–HYB alleles, S10F and S10R,⁹ are indicated by horizontal dotted arrows. The CEL and CELP sequences shown in the Figure correspond to nucleotide positions 12883–13599 and 28652–29371, respectively, from GenBank accession number AF072711.1.

Supplementary Table 1. PCR primer pairs used in this study

Supplementary Table 2. Primers used for sequencing the full-length CEL–HYB1 and CEL–HYB2a transcripts

Abbreviations used in this paper

CP

chronic pancreatitis

NAHR

non-allelic homologous recombination

NMD

nonsense-mediated mRNA decay

RT-PCR

reverse transcription-PCR

SNP

single-nucleotide polymorphism

VNTR

variable number tandem repeat

WT

wild-type