Heterogeneity in Hereditary Pancreatitis

Majed J. Dasouki,1* Joy Cogan,1 Marshall L. Summar,1 Wallace Neblitt III,2 Tatiana Foroud,3Dan Koller,3 and John A. Phillips III1

1Department of Pediatrics, Vanderbilt University, Nashville, Tennessee2Department of Pediatric Surgery, Vanderbilt University, Nashville, Tennessee3Department of Medical and Molecular Genetics, Indiana University, Indianapolis, Indiana

Hereditary pancreatitis (HP) is the mostcommon form of chronic relapsing pancre-atitis in childhood, and may account for∼25% of adult cases with chronic idiopathicpancreatitis. Recently, an arginine-histi-dine (R117H) mutation within the cationictrypsinogen gene was found in 5/5 familiesstudied with HP. In this study we report onthe results of linkage and direct mutationalanalysis for the common R117H mutationexamined in 8 nonrelated families with he-reditary pancreatitis. Two-point linkageanalysis with the 7q35 marker D7S676, doneinitially in 4 families, yielded lod scores thatwere positive in 2, negative in one, andweakly positive in one. Direct mutationalanalysis of exon 3 of the cationic trypsino-gen gene in 6 families showed that all symp-tomatic individuals tested were heterozy-gous for the R117H mutation. Also, severalasymptomatic but at-risk relatives werefound to be heterozygous for this mutation.Affected individuals in the remaining 2families did not have the mutation. Radia-tion hybrid mapping using the Genebridge 4panel assigned the trypsinogen gene tochromosome region 7q35, 2.9 cR distal toETS WI-9353 and 3.8 cR proximal the di-nucleotide repeat marker D7S676. The nega-tive linkage and absence of the trypsinogenmutation in 2/8 families suggest locus het-erogeneity in HP. Analysis of the R117H mu-tation is useful in identifying presymptom-atic ‘‘at-risk’’ relatives and in genetic coun-seling. Also, it can be useful in identifyingchildren and adults with isolated chronicidiopathic pancreatitis. Am. J. Med. Genet.77:47–53, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: hereditary pancreatitis; en-doscopic retrograde cholan-giopancreatography; loga-rithm of odds; expressedtagged sequence; centiray; T-cell receptor b polypeptide;theta (recombination frac-tion); polymerase chain reac-tion

INTRODUCTION

Hereditary pancreatitis (HP), defined as inflamma-tion of the pancreas, usually recurrent from childhood,with autosomal-dominant inheritance, was first de-scribed by Comfort and Steinberg [1952]. Reduced pen-etrance in HP (∼80%) has long been recognized [Sibert,1978]. So far, several hundred cases, including threesets of affected monozygotic twins, have been reportedfrom different parts of the world [Freude et al., 1992].While the exact prevalence of chronic pancreatitis isnot known, a prevalence of 0.04–5% has been sug-gested, with as many as 1

4of these having HP [Steer et

al., 1995]. On the other hand, HP is the most commoncause of chronic relapsing pancreatitis in childhood,presenting as early as infancy but more commonly dur-ing the first decade of life. The frequency and the se-verity of episodes of pancreatitis are unpredictable inHP, and it cannot be differentiated clinically from othercauses of childhood pancreatitis. Diagnosis of HP isusually made after the exclusion of other metabolic dis-orders such as hyperlipidemia, hypocalciuric hypercal-cemia, and hyperparathyroidism in the presence offamily history consistent with dominant inheritance.Serious complications secondary to HP include: pancre-atic exocrine insufficiency, diabetes mellitus, portal hy-pertension, hemorrhagic pleural effusion, addiction tonarcotics, and increased incidence of pancreatic carci-noma [Lowenfels et al., 1993, 1997]. While the elevatedserum amylase and lipase, pancreatic calcification, andduct dilatation seen on abdominal ultrasound and CTscan suggest the diagnosis, endoscopic retrograde chol-angiopancreatography (ERCP) is often needed to con-firm the diagnosis of HP in chronic pancreatitis. Treat-ment is largely symptomatic, with surgery being usedto relieve the pain of recurring acute pancreatitis.

*Correspondence to: Majed J. Dasouki, M.D., Division of Ge-netics, Department of Pediatrics, DD 2205, MCN, VanderbiltUniversity, Nashville, TN 37232. E-mail: majed.dasouki@mcmail.vanderbilt.edu

Received 2 September 1997; Accepted 7 November 1997

American Journal of Medical Genetics 77:47–53 (1998)

© 1998 Wiley-Liss, Inc.

Recently, Le Bodic et al. [1996] and Whitcomb et al.[1996] independently found that HP was linked withtwo 7q35 markers (D7S661 and D7S676) in 2 largeunrelated families examined by microsatellite DNAmarkers in a wide-based genome search. Whitcomb etal. [1996] considered the T-cell receptor and serine pro-tease-1 as the HP candidate genes and excluded thecarboxypeptidase A1 (CPA1) gene locus, as it mappedcentrometric to HP. By direct sequencing of the exonfor the cationic trypsinogen gene, Whitcomb et al.[1996] then identified a histidine substitution for argi-nine (R117H). In this report, we report on our linkageand mutation analyses of 8 additional kindreds, andalso report the results of radiation hybrid mapping ofthe trypsinogen gene to identify additional markers forlinkage and genetic evidence of heterogeneity in twoHP kindreds.

MATERIALS AND METHODSPedigree Identification

Eight unrelated kindreds with HP were identifiedthrough the Pediatric Surgery and GastroenterologyServices at Vanderbilt University Medical Center. Out-of-state family relatives were interviewed by telephoneand their primary care providers were contacted. In-formed consent was obtained from all participants and/or their parents. Whole blood (10–20 ml) was collectedin EDTA-anticoagulated tubes. Genomic DNA was iso-lated using standard methods. Pedigrees were con-structed using the CYRILLIC program (Fig. 1).

Cytogenetic Analysis

Propositus IV-1 (family A) had chromosomal analysisdone on amniotic fluid because of his mother’s ad-vanced age at the time of amniocentesis. This was fol-lowed by peripheral blood chromosomal studies on hisparents, maternal grandmother, and maternal aunt,using standard cytogenetic methods.

Microsatellite Genotyping and Linkage Analysis

PCR primers for the D7S676 and D7S661 fluorescentdinucleotide microsatellites were labeled with HEXand TET, respectively (Research Genetics, Huntsville,AL). PCR amplification reactions were done in a 25-mlvolume containing: 40 ng genomic DNA, 12.5 ml PCRbuffer (2.5 U Taq polymerase, 0.2 mM each dNTP, 10mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2), and 0.5ml of each primer (8 mM). Cycling was done on an MJRthermal cycler using the following conditions: 94°C ×10 min, then 35 cycles of 94°C × 30 sec, 56°C × 30 sec,and 72°C × 30 sec, followed by a final extension step of72°C × 10 min. The fluorescently labeled products werethen electrophoresed on a 377 ABI Applied BiosystemsInstrument DNA sequencer (Foster City, CA). An in-ternal size standard (TAMARA 500™), GENESCAN™672, and Genotyper™ software were used to automati-cally assign the allele sizes. Two-point lod scores(LODs) between the segregation of the HP phenotypein families A–D and the D7S661 and D7S676 loci weregenerated using the MLINK program of the LINKAGEpackage (Version 5.03). An autosomal-dominant inher-

itance pattern, 65% and 80% penetrances at below andabove age 20 years, respectively, and a disease allelefrequency of 0.0001, were assumed for these calcula-tions. In addition, the marker allele frequencies wereestimated using the USER M13 subroutine of theMENDEL package. This calculates the maximum like-lihood estimates of the allele frequencies in the popu-lation from which the pedigrees were drawn, using theobserved alleles in the genotyped sample and condi-tioning on the pedigree structures therein.

Tryspsinogen Exon 3 Mutation Analysis

The forward (U306) and reverse (L1197) primers re-ported by Whitcomb et al. [1996] were used. The PCRamplification conditions were optimized using the In-vitrogen Optimization Kitt (Carlsbad, CA). PCR am-plifications were carried out in 50-ml volumes contain-ing: 2 ml of genomic DNA (50 ng), 10 pmol of forwardand reverse primers, 10 ml 5 × buffer (pH 8.5), 300 mMTris-HCl, 75 mM (NH4)2SO4, 2.5 mM MgCl2, 5 ml of 10mM dNTPs, and 0.5 ml (2.5 units) of AmpliTaq Gold(Perkin Elmer). Cycling conditions were: initial dena-turation for 10 min at 95°C, followed by 40 cycles of94°C × 1 min, 58°C × 1 min, 72°C × 2 min, and a finalextension at 72°C for 10 min. Twenty microliters of the911-bp PCR product were digested with AflIII (NewEngland Biolabs), using conditions recommended bythe manufacturer. The undigested and digested PCRproducts were then electrophoresed on 2% (1% Seakemand 1% NuSieve) agarose gels stained with ethidiumbromide (Fig. 2).

Radiation Hybrid Mapping

Radiation hybrid mapping was performed using theGenebridge 4 Whole-Genome Radiation Hybrid Panel[Walter et al., 1994]. Buffer optimization was carriedout using the Stratagene PCR Optimization Kitt (LaJolla, CA). Twenty-five nanograms of genomic DNAfrom each hybrid were used for amplification in a totalvolume of 10 ml containing 5 ml of PCR buffer (20 mMTris-HCL, pH 8.3, 50 mM KCL, 2 mM MgCl2, and 0.1%vol/vol Tween-20), 200 nM of each of the forward andreverse primers, 0.2 units of AmpliTaq Gold (PerkinElmer) DNA polymerase, and 25 mM of each of the fourdNTPs. Cycling conditions were as described above.The PCR products were mixed with 3 ml of loadingbuffer (50 mM EDTA, 0.5% SDS, 25% glycerol, and0.05% Bromophenol blue) and separated on 1% ethid-ium bromide-stained agarose gel in 1 × TBE buffer. Thescreening results were processed by the RHMAPPERsoftware program at the Whitehead Institute/MIT Cen-ter for Genome Research (Cambridge, MA).

RESULTSPedigree and Cytogenetic Analyses

Of the 98 participants, 37/98 (38%) were known tohave HP. Those affecteds ranged in age from 2–60years. In family A, the propositus (IV-1) and his mother(III-5), who is known to have HP, were both found tohave a balanced translocation: 46,XY,t(5;11)(q13;p15)[Dasouki et al., 1994]. However, the maternal grand-mother (II-5) and maternal aunt (III-12), who also haveHP, had normal [46,XX] karyotypes without this trans-

48 Dasouki et al.

Fig. 1. Pedigrees A–H. Individuals III-5 and IV-1 in family A carry a balanced translocation 5q13;11p15 and the R117H mutation, while IV-2 and IV-3have the HP mutation without the translocation. h/s, normal; l/g, HP; / , DM; / , chromosomal translocation.

Hereditary Pancreatitis 49

Fig. 1. (Continued).

50 Dasouki et al.

location. Propositus IV-10 in family C is known to havepancreatitis and pancreas divisum. No other relative isknown to have this anomaly.

Linkage Analysis

Table I shows the results of two-point lod (LOD)scores for family E between HP and the loci D7S661and D7S676. LODs were negative at both loci in thisfamily, suggesting a different locus for their hereditarypancreatitis. Due to the small family size, linkageanalysis could not be done on family G. Families B–Dhad two-point lod scores of 1.41, 1.24, and 0.03, respec-tively, with D7S676.

PCR Mutational Analysis

The results of direct mutational analysis by PCR areshown in Figure 2. All known affected relatives tested

were positive for the R117H substitution demonstratedby the presence of the AflIII restriction site, except infamilies E and G, which lacked the mutation. In addi-tion, 0/150 chromosomes from 75 unrelated individualsnot known to have HP were also negative for this re-striction site, making it very unlikely to be a DNA poly-morphism. In family A, individual IV-3 and hisbrother, who are at risk for inheriting the (5q13;11p15)translocation, had normal chromosomes, while theyboth inherited the R117H mutation from their affectedmother. The obligate carrier (IV-13) in family H, and 3(undiagnosed) first- and second-degree relatives (IV-9,V-5, and V-7), were also found to have this mutation.Individuals II-8 (obligate carrier) and III-11 in familyB, known to have diabetes mellitus, also had the mu-tation. In family C, the asymptomatic individuals II-4,III-7, III-22, and III-26 were found to have this muta-tion, while individual III-14, who has a history of ab-dominal pain and whose sister (III-13) and motherhave HP, did not have the mutation.

Radiation Hybrid Mapping

Honey et al. [1984] mapped the human trypsinogen(serine protease-1) gene to chromosome region 7q22–ter, using somatic cell hybridization. The assignment ofthis gene to 7q35 is supported by finding its sequencewithin the sequence of the T-cell receptor beta-chain(TCRb) gene [Rowen et al., 1996], and by the linkagebetween microsatellite markers in this region and HP[Le Bodic et al., 1996; Whitcomb et al., 1996; Pandya etal., 1996]. Using radiation hybrid analysis, we foundthat the trypsinogen gene is 3.8 centirays (cR) proximalto the dinucleotide marker D7S676, and 2.9 cR distal tothe EST WI-9353.

DISCUSSION

The hypothesis that pancreatitis may result from in-appropriate activation of pancreatic proenzymes wasfirst considered by Chiari [1896]. Trypsin is the triggerenzyme solely responsible for activating all other pan-creatic zymogens. Therefore, a defect in this moleculecould conceivably result in destruction of the pancreas,leading to pancreatitis. The trypsin precursors, tryp-sinogen 1 (anionic), trypsinogen 2 (mesotrypsinogen),and trypsinogen 3 (cationic), are normally activated bythe duodenal enzyme enterokinase (enteropeptidase).Human trypsinogens, especially the cationic, autoacti-vate more readily than those of other species, depend-ing on the pH and calcium concentration of pancreaticjuice [Armando Madrazo-de la Garza et al., 1993]. Au-toactivation and enzymatic activation of trypsinogengenerate trace amounts of active trypsin within pan-

Fig. 2. Agarose (1% Seakem and 1% NuSieve) gel electrophoresis ofPCR products of cationic trypsinogen gene. Fragment sizes of the molecu-lar weight marker shown in lanes 1 and 10 are: 1,000, 700, 525, 400, 300,200, and 100 base pairs. PCR products were all digested with the restric-tion enzyme AflIII. Normal alleles are represented by the 911-bp fragment,while the mutant allele is shown as two smaller fragments (565 and 346 bp).Individuals in lanes 4, 7, and 12 are known to have HP.

TABLE I. Linkage Analysis Between HP and 7q35 Markers inFamily E*

Marker

u

0.0 0.01 0.05 0.1 0.2 0.3 0.4

D7S676 −2.88 −0.9 −0.27 −0.06 0.08 0.10 0.07D7S661 −5.11 −3.11 −1.72 −1.14 −0.58 −0.30 −0.12

*u, recombination fraction between HP and the marker.

Hereditary Pancreatitis 51

creatic acinar cells. Normally, trypsin is inactivated bybinding to the pancreatic secretory trypsin inhibitor(PSTI, Kazal type). In their X-ray crystallographicanalysis of the cationic trypsinogen, Whitcomb et al.[1996] predicted that the arginine 117 histidine substi-tution would disrupt a trypsin-sensitive site, leading tocontinuous activation of tryopsinogen and other zymo-gens (mesotrypsin and enzyme Y), causing autodiges-tion and pancreatitis. Mapping the cationic trypsino-gen gene within the TCRb gene cluster raises somequestions about a possible interaction between the twoloci. Genotyping of the TCRb locus in complex autoim-mune disorders showed that it may contain gene seg-ments that may confer susceptibility to such disorders.In HLA-DR4-positive rheumatoid arthritis patients,strong linkage disequilibrium was found between 3TCRbV (BV6S7*1, 13S5P*1, and 12S4*2) gene seg-ments, suggesting genetic susceptibility to rheumatoidarthritis through an interaction with HLA-DR4 [Mu etal., 1996]. Also, Eppen et al. [1997] showed a stronggenetic predisposition to multiple sclerosis in individu-als with the HLA-DRB*03+ and the TCRbV6S3 allele.The human PSTI gene was also found to have interleu-kin-6 (IL-6) response elements between Kb −3.84–−3.8[Yasuda et al., 1993]. Therefore, through a complex in-teraction between TCRb, PSTI, and possibly other loci,penetrance of R117H may be altered.

We found that 2/8 (25%) of our HP families did nothave the R117H mutation. The absence of this muta-tion in both families, and the negative LOD scores infamily E at both loci, strongly suggest heterogeneity.So far, the R117H mutation has been found in 11/13(85%) families with HP. If the families reported by LeBodic et al. [1996] and Pandya et al. [1996] are in-cluded, then the frequency of this mutation becomes87%. Matthew et al. [1994] recommended that ERCPbe performed in all children with chronic pancreatitisto outline ductal anatomy and to aid in establishing adiagnosis. Alternatively, this mutational assay couldbe used to study patients with chronic pancreatitiswith or without family history, to establish the diagno-sis without ERCP. In addition, children with chronicrecurrent abdominal pain suggestive of pancreatitismight also benefit from this approach. The recent dis-covery of two novel mutations in the cystic fibrosistransmembrane conductor regulator (CFTR) gene in 2hereditary pancreatitis nonrelated families supportsour finding of genetic heterogeneity [Ravnik-Glavac etal., 1996]. Both mutations (L327R and V1190P) segre-gated with the disease within these families. Recently,Gorry et al. [1997] identified 2 new families with he-reditary pancreatitis that demonstrated an asparaginesubstitution by isoleucine (N21I) in exon 2 of the cat-ionic trypsinogen gene. The possibility of a mutationwithin exon 2 (N21I) of the cationic trypsinogen gene orthe CFTR gene in families E and G is under investiga-tion.

Pancreas divisum is the most common congenitalanomaly of the pancreas, resulting from incomplete fu-sion of the dorsal and ventral pancreatic ductal sys-tems. It may be associated with stenosis of either theaccessory or main duodenal papilla, as seen by ERCP.As many as 50% of patients investigated by ERCP for‘‘idiopathic’’ pancreatitis were found to have pancreas

divisum [Bernard et al., 1990; Cotton, 1980]. Beggs andSalmon [1984] reported the coexistence of hereditarypancreatitis and pancreas divisum, and recently,Muzaffar et al. [1996] reported what looks like the firstexample of familial pancreas divisum and HP. In ourgroup of patients, only patient IV-10 in family C hadsuch an association. No other relative was known tohave this anomaly. Whether the common HP mutationis or is not frequent in patients with pancreas divisumwill be important in determining the possible relation-ship between these two disorders.

In summary, this simple PCR assay for the cationictrypsinogen gene provides a simple method for con-firming the HP diagnosis in most patients and their‘‘at-risk’’ family members. The degree of genetic hetero-geneity can be better assessed by examining morefamilies with HP. Further studies will be needed toexamine the possible role for the HP gene in a varietyof conditions, such as idiopathic pancreatitis, pancreasdivisum, chronic recurrent abdominal pain, and pan-creatic carcinoma, since the risk of malignancy in pa-tients with HP is increased [Lowenfels et al., 1993].

ACKNOWLEDGMENTS

The authors sincerely thank all the family memberswho participated in this study. We thank Drs. BrianReidel, Karen Crissinger, and Fayez Ghishan for refer-ring their patients. We also acknowledge Dr. M.R.S.Krishnamani’s work with the radiation hybrid panels.

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