Original Articles
 

By Dr. Arnaud Boulling , Dr. Jian M Chen , Dr. Isabelle Callebaut , Dr. Claude Férec
Corresponding Author Dr. Jian M Chen
INSERM U1078 and EFS-Bretagne, - France 29218
Submitting Author Dr. Jian-min Chen
Other Authors Dr. Arnaud Boulling
INSERM U1078 and EFS-Bretagne, - France

Dr. Isabelle Callebaut
UMR 7590 CNRS-University Pierre et Marie Curie, - France

Dr. Claude Férec
INSERM U1078 and EFS-Bretagne, - France

GENETICS

Chronic pancreatitis, Expression plasmid, Functional analysis, Minigene, Missense mutation, Molecular modeling, Pancreatic secretory trypsin inhibitor, PSTI, Quantitative RT-PCR, SPINK N34S haplotype

Boulling A, Chen JM, Callebaut I, Férec C. Is the SPINK1 p.Asn34Ser Missense Mutation Per se the True Culprit within its Associated Haplotype?. WebmedCentral GENETICS 2012;3(2):WMC003084
doi: 10.9754/journal.wmc.2012.003084

This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
No
Submitted on: 24 Feb 2012 04:07:04 PM GMT
Published on: 25 Feb 2012 08:47:42 AM GMT

Abstract


Although SPINK1, which encodes the pancreatic secretory trypsin inhibitor (PSTI), has been firmly established as a chronic pancreatitis-predisposing gene, the causal variant within the most common c.101A>G (p.Asn34Ser)-containing haplotype remains to be identified. The low penetrance of this haplotype implies a minor effect on gene expression and/or protein function; such a minor effect may not have been readily detectable in the previous studies. Here, we focused on the putative functional effect of the p.Asn34Ser haplotype on pre-mRNA splicing and mRNA stability. Two measures were taken to maximize the relevance and accuracy of our in vitro analysis. First, all five cis-linked variants were analyzed together within a single expression plasmid. Second, a real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay with high accuracy and reproducibility was developed. The exclusion of a minor effect of the p.AsnN34Ser haplotype on gene expression prompted us to reconsider the possible role of the p.Asn34Ser missense mutation in trypsin inhibition. A survey of previous studies that analyzed the inhibitory activity of purified recombinant wild-type and p.Asn34Ser mutant PSTI peptides revealed that a minor effect of the p.Asn34Ser missense mutation on trypsin inhibition appeared to be overlooked. Moreover, the reduced inhibition of trypsin by the p.Asn34Ser mutant peptide is consistent with findings from analyses of PSTI’s 3D structures and with the presence of an increase in p.Asn34Ser mutants in the urine of p.Asn34Ser heterozygotes. A combination of these diverse lines of evidence strongly suggests that the p.Asn34Ser missense mutation is the true culprit within its associated haplotype. The clarification of this issue is pivotal for developing targeted therapies for pancreatitis.

Introduction


Chronic pancreatitis is a progressive inflammatory disease in which pancreatic secretory parenchyma is destroyed and replaced by fibrous tissue, eventually leading to the impairment of both exocrine and endocrine functions (Braganza et al. 2011). Although SPINK1, which encodes the pancreatic secretory trypsin inhibitor (PSTI), was firmly established as a chronic pancreatitis-predisposing gene, a key question pertaining to the causal variant within the c.101A>G (p.N34S)-containing haplotype (from now on termed N34S haplotype; Illustration 1) remains to be answered (Chen and Férec 2009). The N34S haplotype is the most common SPINK1 haplotype associated with chronic pancreatitis; a meta-analytic study demonstrated that it is associated with a 13-fold higher risk for idiopathic chronic pancreatitis (allele frequency of 9.2% in patients versus 0.7% in controls) and an approximately 5-fold higher risk for alcoholic chronic pancreatitis in Caucasians (Aoun et al. 2008). It is also significantly associated with tropical chronic pancreatitis (reviewed in (Mahurkar et al. 2009; Witt and Bhatia 2008)). Based upon the role of SPINK1 in the etiology of chronic pancreatitis, the N34S haplotype undoubtedly results in a loss-of-function effect. The coding c.101A>G alteration (the other four variants in cis are intronic; Illustration 1) was initially assumed to be the causal variant; the resulting missense mutation, N34S, is located near the Lys41–Ile42 reactive bond and thus may impair PSTI’s inhibitory capacity (Witt et al. 2000). In support of this hypothesis, both molecular modeling and secondary structure prediction suggested that N34S could induce significant structural changes that may weaken the binding of PSTI to trypsin (Kuwata et al. 2003; Pfützer et al. 2000). However, N34S per se was reported not to affect either PSTI secretion/expression or trypsin inhibition activity in three in vitro studies (Boulling et al. 2007; Király et al. 2007; Kuwata et al. 2002). Alternative mechanisms such as aberrant splicing due to the intronic variants (Chen et al. 2001) were also proposed. However, reverse transcriptase-PCR analysis of total RNA isolated from surgically resected pancreatic tissues of two N34S homozygotes did not reveal any aberrantly spliced SPINK1 transcripts (Masamune et al. 2007). More recently, the putative effect of the four intronic variants on mRNA splicing was evaluated in a minigene system (Kereszturi et al. 2009). Again, no detectable functional effect was reported.
Taking the above functional analytic data (Boulling et al. 2007; Kereszturi et al. 2009; Király et al. 2007; Kuwata et al. 2002; Masamune et al. 2007) together, the Sahin-Tóth group proposed that the true pathogenic variant within the N34S haplotype is likely located in the thus far uncharacterized flanking regions of the SPINK1 gene. Nevertheless, it should be noted that the low penetrance of the most common N34S haplotype implies a minor effect on gene expression or/and protein function; such a minor effect may not have been readily detectable in the previous studies (Chen and Férec 2009). Here, we focused on the putative functional effect of the N34S haplotype on pre-mRNA splicing and mRNA stability. Two measures were taken to maximize the relevance and accuracy of our in vitro analysis. First, all five cis-linked variants were analyzed together within a single expression plasmid. Second, a real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay with high accuracy and reproducibility was developed. The exclusion of a minor effect of the N34S haplotype on gene expression prompted us to reconsider the possible role of the N34S missense mutation in trypsin inhibition.

Materials and Methods


Construction of expression plasmids harboring full-length SPINK1 genomic sequences
An approximately 7 kb fragment containing the full-length SPINK1 genomic sequence (defined here as beginning at the translation initiation codon and ending at the translation termination codon; Illustration 1) was amplified separately from genomic DNAs from a healthy wild-type SPINK1 homozygote (WT) and three SPINK1 N34S homozygotes with chronic pancreatitis. The long-range PCR was performed with 150 ng DNA in a 50 µL reaction mixture containing 2.5 U high fidelity TaKaRa La TaqTM DNA polymerase, 8 µL TakaRa dNTP Mix, and 2 µM each of primer pair C1 (Illustration 2). The PCR program had an initial denaturation at 94°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing/extension at 68°C for 10 min, and a final extension step at 72°C for 10 min. Each of the PCR products was subcloned into the pcDNA3.1/V5-His-TOPO vector (Invitrogen) in accordance with the manufacturer’s instructions.
Construction of a reference minigene
An approximately 1.2 kb fragment containing exon 8, intron 8 and exon 9 of the GP2 gene was amplified using the classic Amplitaq DNA polymerase (Applied Biosystem). NcoI and XbaI restriction sites were incorporated into the forward and reverse primers designed for this purpose (primer pair C2, Illustration 2). The amplified fragment was then used to replace the NcoI/XbaI fragment encompassing the entire luc+ coding sequence of the pGL3-Control Vector (Promega) by directional cloning. The GP2 gene structure is in accordance with our previous report (Masson et al. 2010).
Cell culture, transfection and reverse transcription
Human pancreatic adenocarcinoma (COLO-357) cells (Morgan et al. 1980) were grown under conditions described elsewhere (Boulling et al. 2011). Transfections were carried out as previously described (Boulling et al. 2010; Boulling et al. 2011) using 4 µg of the pcDNA3.1-SPINK1 expression plasmid (for conventional RT-PCR) and 2 µg of the pcDNA3.1-SPINK1 expression plasmid plus 2 µg of the pGL3-GP2 reference minigene (for real-time quantitative RT-PCR). At 48 h after transfection, the cells were harvested for total RNA extraction using an RNeasy Mini kit (Qiagen). The RNA concentration and purity were determined by measuring the OD at 260 nm and 280 nm. Reverse transcription was performed in a 20 µL mixture containing 1 µg RNA, 10 U RNAse inhibitor (Promega), 250 ng OligodT15 (Promega), 500 µM dNTPs and 4 U Omniscript Reverse Transcriptase (Qiagen) at 37°C for 1 h.
Conventional RT-PCR
The effect of the N34S haplotype on pre-mRNA splicing was evaluated by conventional RT-PCR using primers located within the 5’- and 3’-untranslated regions of the pcDNA3.1-SPINK1 expression plasmids (primer pair Q1, Illustration 2). The RT-PCR reaction was performed in a 25-µL mixture containing 1 U Amplitaq DNA polymerase, 200 µM dNTPs, 2 µL cDNA and 0.4 µM of each primer. The PCR program had an initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 45 s, and a final extension step at 72°C for 10 min.
Real-time quantitative RT-PCR
Primers used for specific amplification of the SPINK1 transcripts expressed from the pcDNA3.1-SPINK1 constructs are the same used for the aforementioned conventional RT-PCR. In addition, reference GP2 transcripts were amplified using primers located within the 5’- and 3’-untranslated regions of the co-transfected reference minigene pGL3-GP (primer pair Q2, Illustration 2). Real-time quantitative RT-PCR was performed in a 25-µL mixture containing 12.5 µL of 2X Quantitect SYBR Green Master MIX (Qiagen), 2.5 µL of diluted cDNA and 0.3 µM each of primer pair Q1 or primer pair Q2. The PCR program had an initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.
The relative expression ratio of a SPINK1 expression plasmid is calculated based on its E and crossing point (CP) deviation versus those of a control and is expressed in comparison to the reference GP2 minigene in accordance with Pfaffl’s mathematical model (Pfaffl 2001) (see Results and Discussion). Quantitative PCR reactions were performed three times (each in triplicate) and read in Chromo4 (Biorad). The difference between the expression level of the N34S haplotype and that of the wild-type haplotype was assessed for significance by the Student’s t-test.

Results and Discussion


Combining all five cis-linked SPINK1 variants within a single expression plasmid
In their minigene analysis of the four intronic variants in linkage with c.101A>G (N34S), Sahin-Tóth and colleagues placed single introns in the appropriate context of the SPINK1 cDNA. For example, the first minigene comprised exon 1, intron 1, exon 2, exon 3 and exon 4 of the SPINK1 gene (Kereszturi et al. 2009). To maximize the physiological relevance of the in vitro analysis, we constructed expression plasmids that harbored all of the coding sequences and all three introns of the SPINK1 gene in their appropriate genomic context. To this end, a pair of primers (primer pair C1, Illustration 2) was used to amplify the approximately 7 kb SPINK1 genomic sequence that starts at the translation initiation codon and ends at the translation termination codon (Illustration 1) by means of long-range PCR. The amplicons from a healthy wild-type (WT) SPINK1 homozygote and three SPINK1 N34S homozygotes with chronic pancreatitis were separately subcloned into the pcDNA3.1/V5-His-TOPO vector. The resulting expression plasmids were termed pcDNA3.1-SPINK1-WT, -N34Sa, -N34Sb and -N34Sc.
Conventional RT-PCR for detecting aberrant pre-mRNA splicing
We first investigated if the N34S haplotype could cause aberrant pre-mRNA splicing of the SPINK1 gene by means of conventional RT-PCR. The aforementioned four SPINK1 expression plasmids were transfected into human pancreatic adenocarcinoma (COLO-357) cells in parallel under the same conditions. To avoid amplifying SPINK1 transcripts that were endogenously expressed in the COLO-357 cells, a primer pair that is located within the 5’- and 3’-untranslated regions relative to the pcDNA3.1-SPINK1 expression plasmids was designed (primer pair Q1, Illustration 2) for RT-PCR analysis. No aberrantly spliced transcripts were found in any of the three expression plasmids harboring the N34S haplotype (Illustration 3), which is entirely consistent with the finding from RT-PCR analysis of SPINK1 transcripts in pancreatic tissues surgically resected from two N34S homozygotes (Masamune et al. 2007). However, both analyses are not capable of detecting minor quantitative changes due to altered mRNA stability; a minor aberrantly spliced transcript might not be visible in the agarose gel due to nonsense-mediated mRNA decay, and the correctly spliced transcript containing the c.101A>G substitution may be less stable than the wide-type transcript. The end results of either scenario would be a reduced level of the c.101A>G-containing transcript relative to the wild-type transcript.
Real-time quantitative RT-PCR for accurately determining mRNA expression
To exclude the possibility that the N34S haplotype has a minor effect on the expression level of SPINK1 mRNA, we determined the relative expression ratio of each of the three pcDNA3.1-SPINK1-N34S constructs (defined as the sample target genes in our assay) versus the pcDNA3.1-SPINK1-WT (defined as the control target gene) relative to a co-transfected reference gene (see below for details) by means of real-time quantitative RT-PCR. This was done in accordance with Pfaffl’s mathematical model (Pfaffl 2001; Illustration 4), where Etarget = real-time quantitative RT-PCR efficiency of the target gene transcript; Eref  = real-time PCR efficiency of the reference gene transcript; ?CPtarget = CP (cross point; the point at which the fluorescence rises appreciably above the background fluorescence) deviation of the pcDNA3.1–SPINK1-WT control transcript minus the pcDNA3.1-SPINK1-N34Sa, -N34Sb or -N34Sc transcripts; ?CPref = CP deviation of the reference transcript co-transfected with the pcDNA3.1-SPINK1-WT control transcript minus the reference transcript co-transfected with the N34Sa, -N34Sb or -N34Sc transcripts.
To construct the reference gene, an approximately 1.2 kb fragment containing exon 8, intron 8 and exon 9 of the GP2 gene was amplified from the genomic DNA of a healthy subject using primer pair C2 (Illustration 2). The amplicons were then used to replace the entire luc+ coding sequence of the pGL3-control vector by directional cloning, resulting in the reference minigene pGL3-GP2.
Real-time quantitative RT-PCR requires highly specific primers for both the target and reference genes. The aforementioned primer pair Q1 (Illustration 2) was used for this purpose with respect to the target gene. As for the reference gene, we designed a primer pair located within the 5’- and 3’-untranslated regions relative to the pGL3-GP2 minigene (primer pair Q2, Illustration 2). Using these primer pairs, single bands of desired lengths were amplified from cDNAs prepared from pcDNA3.1–SPINK1-WT- and pGL3-GP2-transfected COLO-357 cells, respectively (Illustration 5). Thus, both primer pairs satisfied the required high specificity.
To establish optimal conditions for real-time quantitative RT-PCR, 2 µg of the pcDNA3.1-SPINK1-WT expression plasmid plus 2 µg of the pGL3-GP2 reference minigene were co-transfected into COLO-357 cells. At 48 h after transfection, the cells were harvested for total RNA extraction, and reverse transcription was performed in a 20-µL mixture containing 1 µg RNA. Four serial dilutions of the resulting cDNA (1:16, 1:64, 1:256 or 1:1024) were then used for real-time quantitative RT-PCR. CP cycles versus cDNA input were then plotted to calculate the slope of the standard curve, and the PCR efficiency was calculated from the slope of the standard curve using the formula: E = 10(–1/slope) (Rasmussen 2001). As shown in Illustration 6, the target SPINK1 and reference GP2 transcripts showed acceptable PCR efficiency rates (E = 1.90 and 1.82, respectively) in the range of the four dilutions with strong linearity (R2 = 0.991 and 0.995, respectively).
We then chose the 1:64 dilution of cDNA for simultaneously assaying the relative expression ratio of pcDNA3.1-N34Sa, -N34Sb and -N34Sc versus the control pcDNA3.1-SPINK1-WT under the aforementioned conditions; we used the reference pGL3-GP2 to normalize the results according to the Pfaffl method. As shown in Illustration 7, the experimentally calculated ratios did not significantly differ from the theoretical ratio of 1, indicating that no significant difference of mRNA expression was found between the N34S haplotype and the wild-type haplotype.
Re-focusing on a defect at the protein level
Having confidently excluded a minor effect of the N34S haplotype on gene expression at the mRNA level, we turned our attention to a potential minor effect of the N34S missense mutation on protein function. To this end, we revisited or reevaluated the current available data in the following three aspects.
Trypsin inhibitory activity of purified PSTI
To date, two studies have analyzed the trypsin inhibitory activity of purified PSTI. The first study expressed the wild-type and N34S mutant PSTI peptides in the host strain Saccharomyces cerevisiae BJ1991. The trypsin inhibitory activity of the purified PSTI peptides was evaluated under five pH conditions. No differences were observed between the wild-type and mutant proteins at pH 5 and pH 8, whereas the mutant proteins showed a slightly lower inhibitory activity at pH 6, 7 and 9 (see Fig. 2 in (Kuwata et al. 2002)). The second study expressed the wild-type and mutant SPINK1 genes in human embryonic kidney 293T cells. Again, the N34S mutant PSTI, either in the secreted conditioned media from transfected cells or as purified PSTI, showed a slight decrease in trypsin inhibitory activity relative to the wild-type molecule (see Figs. 2 and 4 in (Király et al. 2007)). Taken together, these observations provide support to the original hypothesis that the NS4S per se is the causal variant within the common SPINK1 haplotype (Witt et al. 2000).
Examination of 3D structures of human PSTI alone and in complex with enzyme
Although not directly in contact with the enzyme, N34 is located near the enzyme-binding loop, which has been shown from the 3D structures of PTSI in different species to display the flexibility that is required for adaptation to the protease (Bolognesi et al. 1982). The major difference between the structures of human PSTI in an uncomplexed form (pdb 1hpt (Hecht et al. 1992)) and in complex with an enzyme (chymotrypsinogen, pdb 1cgi (Hecht et al. 1991)) lies in the region including N34, which is forced by a rotation of the peptide bond to Y33 of nearly 180°. In the complexed form, the side chain hydroxyl group of Y33 forms a hydrogen bond with T40, whereas in the uncomplexed form, this interaction does not exist (Illustration 8). These conformational dynamics and the dependent Y33-T40 interaction in human PSTI are thus apparently required for the correct formation of the inhibitor-enzyme complex.
In the uncomplexed form of human PSTI (Hecht et al. 1992), N34, the immediate neighbor of Y33, has a noticeable conformation in the left-handed alpha-helical region of the Ramachandran plot (positive ? and ? torsional angles). This conformation is normally only acceptable for glycine and asparagine. In the case of asparagine, the distortion from the ideal peptide geometry is generally stabilized through carbonyl-carbonyl stacking interactions (Deane et al. 1999). Thus, the high-energy conformation at this position, due to the positive ? value, cannot be stabilized when asparagine is mutated to serine. Therefore, the main chain of the N34S variant is likely to adopt another local conformation in this region. This might impact the dynamic behavior of the loop, thereby impairing the required conformational change necessary for forming the Y33-T40 bond and the associated binding to the enzyme.
An impediment of the N34S variant to the formation of the Y33-T40 interaction has been previously postulated by Pfützer and colleagues (Pfützer et al. 2000). Based on the presence of serine at position 34 of the porcine PSTI and the significant structural similarity between human and porcine PSTI peptides, they surmised that the porcine PSTI structure could be a surrogate for the human N34S mutant PSTI structure. Thus, in the human N34S mutant PSTI, the S34 side chain was presumed to point inward toward the protein interior while the Y33 side chain was presumed to point outward (see Fig. 3 in (Pfützer et al. 2000)). This significant structural change would then disrupt the strong interaction between Y33 and T40 of the human PSTI. Nevertheless, it seems risky to use the porcine PSTI structure to speculate on the conformation of the human N34S mutant’s enzyme-binding loop in complex with the enzyme. This is because the human and porcine sequences have differences that could lead to other local conformation(s). In particular, the amino acid at residue 33 of the porcine wild-type PSTI is different from the amino acid at residue 33 in its human counterpart (threonine and tyrosine, respectively).
Surprising enrichment of the N34S variant form in the urine of heterozygous carriers
Using immunoaffinity purification and mass spectrometry, the Stenman group succeeded in differentiating the N34S mutant PSTI from the wild-type peptide in the urine of N34S heterozygous patients with pancreatitis (Valmu et al. 2006). Importantly, they found that the amount of the N34S mutant peptides is consistently higher than that of the wild-type peptide in all seven N34S heterozygous carriers. They suggested two theories to account for this phenomenon. First, the N34S mutant is more stable than the wild-type molecule. Second, wild-type PSTI was more often in a complex with trypsin in urine and thus less detectable by their method relative to the N34S mutant (Valmu et al. 2006). In other words, the wild-type PSTI has a higher binding affinity for trypsin than the N34S mutant. In line with the previous arguments, we favor the second theory over the first one.

Conclusions


As stated by Sahin-Tóth and colleagues, “The mechanism of action of the p.N34S-associated haplotype remains one of the most intriguing, unsolved questions of pancreas genetics” (Kereszturi et al. 2009). In this study, we have provided further evidence that the N34S haplotype is indeed not associated with any functional defect at the mRNA level. In contrast, a survey of previous studies that analyzed the inhibitory activity of purified recombinant wild-type and N34S mutant PSTIs revealed that a minor effect of the N34S missense mutation on trypsin inhibition appeared to have been overlooked (Király et al. 2007; Kuwata et al. 2002). Moreover, a reduced inhibitory activity for trypsin of the N34S mutant peptide is consistent with insights generated from the examination of PSTI’s 3D structures and the presence of increased N34S mutants in the urine of N34S heterozygotes (Valmu et al. 2006). Combining these diverse lines of evidence strongly suggested that the N34S missense mutation is the true culprit within its associated haplotype. Clarification of this issue is pivotal for developing targeted therapies for pancreatitis.

References


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Source(s) of Funding


This work was supported by the INSERM (Institut National de la Santé et de la Recherche Médicale) and the Programme Hospitalier de Recherche Clinique (PHRC R 08-04), France.

Competing Interests


The authors are not aware of any competing interests.

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