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Peptic ulcer inheritance in patients with elevated serum pepsinogen group A levels and without infection of Helicobacter pylori
ALIMENTARYTRACT
916EST LIVER DIS 2000;32:12-9
Peptic ulcer inheritance in patients with elevated serum pepsinogen group A levels pylori and without infection of Helicobacter T. Del Bianco R. Borgonil P. Del Biancol l? Cedar0 F. Vianello G.A. Danieli* F. Di Mario
Background. Peptic ulcer has multifactorial aetiology including genetic factors. We have identified a family with pepsinogen Group A levels. higher than normal, with a high prevalence of ulcer disease and a low prevalence of Helicobacter pylori infection. Aims. Performing linkage analysis in the identified family Patients and Methods. We examined the segregation of pepsinogens with microsatellite dinucleotide repeat DNA markers along chromosome I 1 (DllS480, PYGM] for pepsinogen Group A and along chromosome 6 [DSSIOS, D6SI 6 IO, TRMI] for pepsinogen Group C. Results. In markers examined along chromosome 1 I, linkage analysis provided no evidence for significant causal mutation but, controlling for some risk factors we observed that the probability of falling ill, increases. The linkage analysis along chromosome 6 for pepsinogen Group C did not show a uniform genetic profile. Conclusions. This study evaluates the hypothesis of peptic ulcer inheritance at least in a small group of patients without the common risk factors.
Digest
Liver
Key words:
Ois 2000;32:12-19 heredity;
hyper-pepsinogen
group
A; linkage
analysis;
peptic
ulcer
Introduction Peptic ulcer is a disease of multifactorial aetiology, including genetic factors, life-style factors, and infection with Helicobacter pylori (H. pylori). Peptic disease is caused by, and its persistence is due to the breakdown of a supposed equilibrium between aggressive and defensive factors ’ 2. H. pylori infection is widely accepted to be the predominant cause of chronic gastritis, which is strongly associated with gastric ulcer, and duodenal ulcer 3-5.The putative pathogenic properties of this bacterium may be produced by disease-inducing factors that cause adverse pathological effects in the gastric mucosa, such as pepsinogen stimulation, reduced mucus secretion, increased gastric secretion and others 6. Nevertheless, family and twin studies have demonstrated that peptic ulcer disease is inherited as a multifactorial trait 7-9 and polygenic inheritance has become the prevailing hypothesis for the genetic basis of duodenal ulcer lo. An elevated serum pepsinogen A (PGA) level is inherited as an autosomal-dominant trait in some families with a prominent history of duodenal ulcer “-14. PGA has five electrophoretic isozymogens (PG 1-5) and
12
T. Del Bianco et al.
Peptic ulcer inheritance
is remarkably heterogeneous. This is shown by an extensive protein electrophoretic polymorphism resulting in multiple haplotypes containing different combinations of the individual PGA genes and one or more post-traslational modifications of the primary gene products I5 16. Localisation of the human PGA gene cluster is in chromosome 11, in the region ql lpter I’. The other human pepsinogen: pepsinogen C (PGC) has two electrophoretic isozymogens (PG 6 and 7) I2 13. No genetic variation has been described at protein level, and the localisation of human PGC gene is chromosome 6 in the p21.1-pter region “. It has been reported that the concordance rate for peptic ulcer in monozygotic twins is greater than in dizygotic twins. Since the concordance was less than lOO%, the values could also be interpreted as indicating interactions between genetic predisposition and environmental factors 19.A number of inherited characteristics, such as blood groups and HLA antigens, have been studied to determine the degree of association with peptic ulceration 7 20-23.Recent studies have demonstrated that, in Japanese patients, there is genetic polymorphism of the PGC gene and gastric body ulcer, but not for the haplotype of PGA. The genetic predisposition is not associated with H. pylori infection 2425. The Indian population presents a higher frequency of the two fractions of PGA, that are found to be significantly associated with duodenal ulcer. All the genes of the multiple complex controlling pepsinogen polymorphism seem to be interacting, thereby leading to this association 26. Difficulties have been encountered in attempting to identify the genes involved in the pathogenesis of the disease because most diseases and traits, such as peptic ulcer, heart disease, diabetes, cancer and others, do not follow simple inheritance patterns. The term complex trait refers to any phenotype that does not exhibit classic mendelian recessive or dominant inheritance attributable to a single gene locus. In general, complexities occur when the simple correspondence between genotype and phenotype breaks down, either because the same genotype can result in different phenotypes (due to the effects of change, environment, or interactions with other genes) or different genotypes can result in the same phenotype. It is often impossible to find a genetic marker that shows perfect cosegregation with a complex trait. For this reason, there are many problems that can hamper genetic dissection of complex traits. It is necessary to look for phenotype definition, identifying informative families for genetic studies. Focusing on a highly restricted population may also offer advantages for eventual positional cloning, because one may be able to exploit linkage; ideally families should be selected on the basis of a priori considerations. Previously we
studied a family in a geographically and historically isolated valley of Northeast Italy with a high prevalence of peptic ulcer 27. In this family, PGA levels were higher than normal reference values, as suggested by Rotter et al. ‘I. In agreement with previous reports, these data show that PGA may be considered as a pathophysiological factor associated with development of peptic ulcer, and support the hypothesis of heredity of this factor. The aim of the present study was to perform linkage analysis in the previously studied family with high serum PGA levels and high prevalence of peptic disease and to compare these findings with those from other families with high prevalence of ulcer disease but not with high serum PGA levels, investigating in the same groups the prevalence of H. pylori infection.
Patients and methods Family 1 is the family, previously studied, with a high prevalence of peptic disease living in the isolated valley of Northeast Italy (Val di Resia). The human pedigree consisted of 27 family members in four generations (Fig. 1). Family 2 is another family with a high prevalence of peptic disease living in the Val di Resia but not with high serum PGA levels (Fig. 2). Families 3 and 4 are families living in the same valley but with no history for ulcer disease. The human pedigrees of Families 5 (Fig. 3) and 6 are members living in a nearby valley, not isolated historically and geographically, with, respectively, a history or no history of
Iv
13
Family 2
II 5
6
7
fl-0 8
0
10
11
R3. 2. Family 2 pedigree, with high prevalence of peptic disease but Nithout high serum PGA levels, living in the Val di Resia. For explaiation: see legend Figure I.
peptic disease. Figures l-3 show the only pedigrees of the families with peptic disease. The families were identified through general practitioners working in the area. The diagnosis of peptic disease was performed by upper gastrointestinal endoscopy, X-ray barium meal or at surgery. All the individuals selected for the investigation were previously submitted to studies on the upper gastrointestinal tract and peptic disease was demonstrated. We have also considered the genetic transmission model to explain the inheritance of a disease in pedigrees. All subjects were informed about the study by letter and invited to participate. This study was performed according to the principles of the Declaration of Helsinki, and consent was obtained from each subject after full explanation. In family 1 -80% of the subjects agreed to take put,
Family5
I
8
8
in the other families all participated in the study. For each subject, we used a structured questionnaire for lifestyle factors such as smoking and alcohol intake. In particular, we investigated the use of antisecretory therapy, non-steroidal anti-inflammatory drugs (NSAIDs) and antimicrobial drugs over the previous month. Subjects using antisecretory therapy, NSAIDs and antimicrobial drugs during the last month were excluded from the study to allow the necessary washout period for drug assumption. We also excluded those patients, in Family 1, that have been treated with eradication therapy for H. pylori infection. Blood samples were taken at the time of the medical interview and stored for linkage analysis and to determine serum PGA and PGC levels, gastrin levels, H. pylori antibodies, hepatitis A (HAV) antibodies and blood groups. The method available to localize the gene of the disease is linkage analysis of the cosegregation. Linkage analysis can also be applied when penetrance in unknown and can also be extended to situations in which two or more genes play a role in the inheritance of a disease 28. We have examined the segregation of PGA and PGC with microsatellite dinucleotide repeat DNA markers along chromosome 11 and 6. Since PG genes are localized to the centromere of the human chromosome 11 in the region p I 1+q13 I5 I7 and in the region p2 1.1 of human chromosome 6 Ix, we studied the cosegregation with ulcer disease analyzing the border on markers. These markers were Dl lS480 and PYGM for chromosome 11, D6S 105, D6S1610 and TRMI for chromosome 6. Genomic DNA was extracted according to the salting out procedure. DNA samples were analysed by polymerase chain reactions (PCR). The PCR were performed as follows: 50 ng of genomic DNA was used as template with 10 pM each oligonucleotide primer, 0.4 units of Taq polymerase, 67 mM Tris-HCl (pH 8.8), 16.6 mM ammonium sulphate, 1.5 mM MgCl 2, 0.01% Tween 20, and dGTP, dATP, dTTP, and dCTP each at 100 pM in a final reaction volume of 12 pl. The samples were over-layed with 15 pl of mineral oil to prevent evaporation. PCRs were performed in an MJ Research microplate thermocycler under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The cycle was repeated 30 times. After PCRs, 2~1 aliquots of the reaction mixture were denatured and separated on a 9% denaturing polyacrylamide gel. Gels were then silver stained and dried. Serum PGA and PGC concentrations were determined by PGA and PGC RIA kit (Sorin Biomedica, Saluggia, Italy); the limits of detection were 30-100 pg/l and 2-14 pg/l for PGA and PGC, respectively. The serum gastrin levels were also determined by a
10 11 13 12
Fi3.3. Family 5 pedigree, living near the Val di Resia, with history of peptic disease. For explanation: see legend Figure I.
T. Del Bianco et al.
RIA kit to (Sorin Biomedica, Saluggia, Italy); the normal range established being 0- 115 rig/l. The presence of anti-H. pylori IgG antibodies was determined by an in-house, enzyme linked immunoassay (RADIM, Pomezia, Italy); a threshold titre >20 U/l was used to discriminate H. pylori positive from H. pylori negative individuals. Sera were tested also by ELISA for anti-HAV (Abbott, Campoverde, Italy); the results were expressed in positive or negative for HAV infection. ABO blood groups were determined by a standard haemagglutination assay performed with a 3% suspension of peripheral blood. Linkage analysis was performed using a computer programme named LINKAGE (version 5.1) which estimates the lod-score [an acronym for logarithm of the log odds ratio (OR)], which is the ratio
Fmnlly 2
log10 = LP) L; J--l~ where L(8) is the likelihood function of the samp,c and 8 is the maximum likelihood estimator of the recombination fraction. In practice, lod-scores are evaluated at a limited number of points OS&‘/Z and the value that maximises the lod-score is chosen as the estimate of 19.A lod-score was calculated by assuming 0.7 and 0.9 penetrance. A lod-score of 3 or greater is usually taken as significant evidence of linkage; a lod-score of -2 or lower is taken as evidence of no-association 2y. The association between H. pylori infection and blood group, HAV infection, peptic disease and serum PGC levels was analysed separately using log linear models and OR was used as a measure of the association together with 95% confidence intervals (95% CI) 30.
Results
The serum PGA values show different levels in selected families with peptic disease (Fig. 4). We have studied the serum PGA values and the presence of elevated titres of H. pylori antibodies. If there were high PGA and high H. pylori antibodies, the value of PGA was considered associated with H. pylori infection. We, therefore, began performing linkage analysis in Family 1 because these subjects were found to have peptic disease with high levels of PGA. Furthermore in each family member, serum gastrin levels were normal (data not shown). According to epidemiological studies in the Family considered, there is a
Fig. 4. Serum PGA and anti-H. py/ori levels in families 1, 2 and 5 (see pedigree for references to subjects).
progressive increase in the prevalence of H. pylori infection with age. Even if there is no close correlation between serum PGA levels and serum Anti H. pylori levels, this family is particularly indicated for linkage study because there are several affected individuals who could have inherited the same disease-causing allele from a common ancestor. It is a genetically isolated population. It is possible to exploit information from many historical meioses and, thereby, afford much higher recombinational resolution. In fact, in this analysis, the classical strategy is, first, to detect a major-gene effect by segregation analysis and, second, to seek for linkage with genetic markers by the lod-score method using major-gene parameters. The localisation of the human PGA gene cluster is to the chromosome 11 and, for linkage study, two DNA markers from this region were used. The maximum 15
Peptic ulcer inheritance
kbk I. Lod-score test values at each 0 level for different markers used.
lod score values were -0.049 at eO.4 (70% penetrance) and -0.06 at 0=0.4 (90% penetrance) for Dl lS480 DNA marker, and 0.017 at 60.4 (70% penetrance) and 0.031 at eO.4 (90% penetrance) for PYGM DNA marker. In every examined marker the lod-score test was ~3, thus linkage analysis provided no evidence for significant causal mutations of PGA genes (Table I). The lod-score is not equal or lower than -2 so that linkage analysis cannot accept any association. Of the 20 patients with peptic ulcer disease, eight subjects had never used NSAIDs and four subjects (1114,1115,1116,11123) do not have H. pylori infection. In order to gain a better insight into the genotype risk, the odds of the presence of a small fragment (4.6) (Fig. 5) associated with no H. pylori infection was estimated: 9=
P (fragment
= (4*6)/H.
pylori
= no)
P fragment
*(4-6)/H.
pylori
= no)
We find Y=3, so the probability of having peptic disease for subjects with the same small fragment, without H. pylori infection, is three time greater than the probability of falling ill for a patient without that fragment (Table II). This value, of course, does not support any statistical conclusion because of the very small sample size, however, data show strong evidence of an important effect of the genetic component
4. I. 6meB fragment 46 of chromoeome 11 in ragion ql I-pter. DNA samples were anelysed by poIymerase chain &on. In lane 3, 5. 6 and 8, indicated by arrows, the ekctfwhoretic patterns are identical for the markers amplified from the pwted DNA semples. The fragment 4.6 indicates the haplotype of the disease.
16
I
NW II. Contingency table of fwquertcies between H. p@ri infection and haplotype.
4.6 e4.6
i
3 1
:
4
4
8
on presence of the disease. Moreover, these four patients had elevated values of serum PGA levels. The linkage analysis was performed also for DNA markers along chromosome 6; the results obtained neither exclude nor confirm the association. We also observed that these patients do not show a uniform genetic profile. At this stage of our work, we did not perform the linkage analysis in members of the other families. We thought it better to first find a localisation in the family potentially more informative for disease and then, in other families to test, only the markers near that localised gene. To emphasize our genetic results, we made some statistical considerations on other variables usually considered in the literature. We analyzed the association between H. pylori infection, blood groups, HAV infection and serum PGC levels in Family 1 and in other family members with a history or no history for peptic disease living in this valley and in a nearby valley. We studied a total of 78 subjects. Seropositivity for H. pylori infection is not associated with peptic ulcer disease either Family 1 (p=O.8) or in other families members (p=O.2) even if there is more evidence of independence in data of Family 1. The association between H. pylori infection and blood group 0 is not statistically significant (p=O.504 Family 1 and p=O.533 other families). Furthermore, there is no association between PGC and H. pylori infection (p=O.5 Family 1 and p=O.O6 in other families). As far as concerns the relation between HAV
We III. Analysis of association between H. py/ori infection, blood groups, HAV infection, peptic disease and PGC serum levels in Family 1 and other families members (with peptic disease and control families. Total number of subjects: 781.
LIIT
lbwrlrpir Blood GMUPS vs H. pybri infection HAV infection vs H. pybri infection Peptic disease vs tl. &ori infection PGC vs H. py/wi infection
Family 1 Other families Family 1 Other families Family 1 Other families Family 1 Other families
and H. pylori infection, log-linear analysis shows different results for the two groups: in Family 1 there is independence (p=O.527) while in the other family subjects there would appears to be an association (p=O.Ol; OR=2.208, 95% CI 1.16 to 4.21) (Table III). Discussion In this study, we have identified a family with a high prevalence of peptic ulcer and with high serum PGA levels. Moreover, there is a particular environmental situation. In fact, members living in a valley of Northeast Italy, belong to an ancient barbaric nation, the Ruteni, who, remained isolated, for a long time, from neighbouring communities. Mountains enclose this valley so that, until recent times, there was only one route of access at the bottom of these mountains. The Resiani comprise the most part of the population, only 2.9% of the community members come from other areas. There is a tendency to marry within the same settlement 3’-33. Because of these characteristics, Resiani could, indeed, have formed a rarely genetic isolated found, so they are ideal for genetic studies. The four affected members with no important risk factors show the same fragment of gene localised in the same region to chromosome 11. When in a linkage analysis between a disease and a genetic marker some families show linkage while others do not, then the question often asked is whether the observed variation in the recombination fraction over families is due to change alone or whether it might be due to disease heterogeneity. Linkage is due to the phenomenon that alleles at different loci on a single chromosome are often transmitted together from parent to offspring. On the other hand, even when the loci are physically close, alleles on different homologous chromosomes are sometimes transmitted to one offspring. When the
0.44 0.38 0.39 6.52 0.06 1.49 0.44 3.56
P
OR
i:g 0.52 0.01
2.208
95%lFl
1 .I6421
0.80 0.20 0.50 0.06
latter occurs, a recombination event is said to have separated the two loci 34. In our family, the meiotic events analysed were low because only four subjects have the fragment 4.6, and this situation is not particularly favourable. Since phenotypes are function of the genotypes (our subjects had high serum PGA levels), the probability of the observed phenotypes should be compatible with genotypes 35. Our analysis provided no evidence for significant causal mutations within the region bounded by Dl lS480 and PYGM on chromosome 11. But genetic analysis of complex traits is complicated by many factors: reduced penetrance, aetiologic heterogeneity, the number of loci contributing to disease (and their interactions), mode of inheritance, presence of phenocopies (non-genetic cases), etc. It is still possible, however, that a gene of major effect exists in this region or in another region or in another chromosome, either with low penetrance or with heterogeneity. We think genetic heterogeneity is more likely because the other family members showed no linkage with any of the investigated loci. Some diseases were previously mapped to a chromosome, and other studies had demonstrated that the same disease appears to be transmitted with polymorphic DNA markers by another chromosome 36. Another point is that for most of the models, the use of major gene parameters in lod-score analysis leads to a large bias in estimation of the recombination fraction and sometimes also to a rejection of linkage for the true combination fraction 37. Genetic factors alone, without environmental interactions, could account for this segregation, and vice versa if environmental interactions are important, our sample is ideal because there are subjects with hyper PGA and peptic ulcer and without hyper PGA and peptic ulcer, living in and outside the valley. This study supports the idea of the peptic ulcer inheritance at least in a small group of patients without the common factors of risk such as H. pylori infection and NSAIDs use. So, we are, therefore, encouraged to perform
Peptic ulcer inheritance
linkage analysis in the entire DNA, in our entire sample, to demonstrate or to exclude the heredity. Our hypothesis is also supported by the fact that in Family 1 the data lead to conclusions different from those in the literature. -H. pylori is a very common infection and causes peptic ulcer. In Family 1 H. pylori infection and peptic disease are independent. - Both PGA and PGC are present in the chief cells of the oxyntic glands of the gastric corpus mucosa, but only PGC is present in the gastric antrum. Colonisation of the gastric mucosa by H. pylori is associated with histologically confirmed chronic gastritis. Increased serum PGC concentrations appear to be related to the severity of gastritis in H. pylori infected subjects 38 39. Since peptic ulcer is associated with H. pylori infection, we should find a link also in Family 1 between disease and serum PGC. But as ulcer disease is independent from H. pylori infection, as it is independent from serum PGC levels. - Boren et al. 4” demonstrated that the receptor for H. pylori on the gastric epithelial cell is the blood group antigen Lewis b. Bacteria did not bind to Lewis b antigen substituted with a terminal GalNAcal-3 or Gala1-3 residue (blood group A and B determinants), suggesting that the availability of H. pylori receptors might be reduced in individuals with blood groups A and B compared with those with blood group 0. This speculation seemed to be consistent with the old observation that peptic ulcer is more prevalent in individuals with blood group 0 4’ 42. Recently it was demonstrated that H. pylori adheres to isolated human gastric cells is a manner that is not dependent on Lewis antigen expression on these cells, and expression of Lewis antigens on the surface of gastric cells is not dependent on Lewis antigen expression on erythrocytes 43. In our data there is no evidence of this relationship. - If person-to-person contact plays an important role in H. pylori transmission, the infection seems very similar to that of hepatitis A virus. Some Authors have shown that the seroprevalence of H. pylori infection parallels HAV, suggesting similar modes of transmission 44, others have shown no statistically significant correlation between seroprevalence of H. pylori and HAV 4546. In our results, the relationship between seroprevalences of H. pylori and HAV was not statistically significant in Family 1 whereas it was in the other families. Thus, this study underlines the possible role of the peptic ulcer inheritance (investigated by means of the serum PGA distribution) in patients with a low rate of H. pylori infection located in a rare genetic and environmental isolated situation. 18
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19
916EST LIVER DIS 2000;32:12-9
Peptic ulcer inheritance in patients with elevated serum pepsinogen group A levels pylori and without infection of Helicobacter T. Del Bianco R. Borgonil P. Del Biancol l? Cedar0 F. Vianello G.A. Danieli* F. Di Mario
Background. Peptic ulcer has multifactorial aetiology including genetic factors. We have identified a family with pepsinogen Group A levels. higher than normal, with a high prevalence of ulcer disease and a low prevalence of Helicobacter pylori infection. Aims. Performing linkage analysis in the identified family Patients and Methods. We examined the segregation of pepsinogens with microsatellite dinucleotide repeat DNA markers along chromosome I 1 (DllS480, PYGM] for pepsinogen Group A and along chromosome 6 [DSSIOS, D6SI 6 IO, TRMI] for pepsinogen Group C. Results. In markers examined along chromosome 1 I, linkage analysis provided no evidence for significant causal mutation but, controlling for some risk factors we observed that the probability of falling ill, increases. The linkage analysis along chromosome 6 for pepsinogen Group C did not show a uniform genetic profile. Conclusions. This study evaluates the hypothesis of peptic ulcer inheritance at least in a small group of patients without the common risk factors.
Digest
Liver
Key words:
Ois 2000;32:12-19 heredity;
hyper-pepsinogen
group
A; linkage
analysis;
peptic
ulcer
Introduction Peptic ulcer is a disease of multifactorial aetiology, including genetic factors, life-style factors, and infection with Helicobacter pylori (H. pylori). Peptic disease is caused by, and its persistence is due to the breakdown of a supposed equilibrium between aggressive and defensive factors ’ 2. H. pylori infection is widely accepted to be the predominant cause of chronic gastritis, which is strongly associated with gastric ulcer, and duodenal ulcer 3-5.The putative pathogenic properties of this bacterium may be produced by disease-inducing factors that cause adverse pathological effects in the gastric mucosa, such as pepsinogen stimulation, reduced mucus secretion, increased gastric secretion and others 6. Nevertheless, family and twin studies have demonstrated that peptic ulcer disease is inherited as a multifactorial trait 7-9 and polygenic inheritance has become the prevailing hypothesis for the genetic basis of duodenal ulcer lo. An elevated serum pepsinogen A (PGA) level is inherited as an autosomal-dominant trait in some families with a prominent history of duodenal ulcer “-14. PGA has five electrophoretic isozymogens (PG 1-5) and
12
T. Del Bianco et al.
Peptic ulcer inheritance
is remarkably heterogeneous. This is shown by an extensive protein electrophoretic polymorphism resulting in multiple haplotypes containing different combinations of the individual PGA genes and one or more post-traslational modifications of the primary gene products I5 16. Localisation of the human PGA gene cluster is in chromosome 11, in the region ql lpter I’. The other human pepsinogen: pepsinogen C (PGC) has two electrophoretic isozymogens (PG 6 and 7) I2 13. No genetic variation has been described at protein level, and the localisation of human PGC gene is chromosome 6 in the p21.1-pter region “. It has been reported that the concordance rate for peptic ulcer in monozygotic twins is greater than in dizygotic twins. Since the concordance was less than lOO%, the values could also be interpreted as indicating interactions between genetic predisposition and environmental factors 19.A number of inherited characteristics, such as blood groups and HLA antigens, have been studied to determine the degree of association with peptic ulceration 7 20-23.Recent studies have demonstrated that, in Japanese patients, there is genetic polymorphism of the PGC gene and gastric body ulcer, but not for the haplotype of PGA. The genetic predisposition is not associated with H. pylori infection 2425. The Indian population presents a higher frequency of the two fractions of PGA, that are found to be significantly associated with duodenal ulcer. All the genes of the multiple complex controlling pepsinogen polymorphism seem to be interacting, thereby leading to this association 26. Difficulties have been encountered in attempting to identify the genes involved in the pathogenesis of the disease because most diseases and traits, such as peptic ulcer, heart disease, diabetes, cancer and others, do not follow simple inheritance patterns. The term complex trait refers to any phenotype that does not exhibit classic mendelian recessive or dominant inheritance attributable to a single gene locus. In general, complexities occur when the simple correspondence between genotype and phenotype breaks down, either because the same genotype can result in different phenotypes (due to the effects of change, environment, or interactions with other genes) or different genotypes can result in the same phenotype. It is often impossible to find a genetic marker that shows perfect cosegregation with a complex trait. For this reason, there are many problems that can hamper genetic dissection of complex traits. It is necessary to look for phenotype definition, identifying informative families for genetic studies. Focusing on a highly restricted population may also offer advantages for eventual positional cloning, because one may be able to exploit linkage; ideally families should be selected on the basis of a priori considerations. Previously we
studied a family in a geographically and historically isolated valley of Northeast Italy with a high prevalence of peptic ulcer 27. In this family, PGA levels were higher than normal reference values, as suggested by Rotter et al. ‘I. In agreement with previous reports, these data show that PGA may be considered as a pathophysiological factor associated with development of peptic ulcer, and support the hypothesis of heredity of this factor. The aim of the present study was to perform linkage analysis in the previously studied family with high serum PGA levels and high prevalence of peptic disease and to compare these findings with those from other families with high prevalence of ulcer disease but not with high serum PGA levels, investigating in the same groups the prevalence of H. pylori infection.
Patients and methods Family 1 is the family, previously studied, with a high prevalence of peptic disease living in the isolated valley of Northeast Italy (Val di Resia). The human pedigree consisted of 27 family members in four generations (Fig. 1). Family 2 is another family with a high prevalence of peptic disease living in the Val di Resia but not with high serum PGA levels (Fig. 2). Families 3 and 4 are families living in the same valley but with no history for ulcer disease. The human pedigrees of Families 5 (Fig. 3) and 6 are members living in a nearby valley, not isolated historically and geographically, with, respectively, a history or no history of
Iv
13
Family 2
II 5
6
7
fl-0 8
0
10
11
R3. 2. Family 2 pedigree, with high prevalence of peptic disease but Nithout high serum PGA levels, living in the Val di Resia. For explaiation: see legend Figure I.
peptic disease. Figures l-3 show the only pedigrees of the families with peptic disease. The families were identified through general practitioners working in the area. The diagnosis of peptic disease was performed by upper gastrointestinal endoscopy, X-ray barium meal or at surgery. All the individuals selected for the investigation were previously submitted to studies on the upper gastrointestinal tract and peptic disease was demonstrated. We have also considered the genetic transmission model to explain the inheritance of a disease in pedigrees. All subjects were informed about the study by letter and invited to participate. This study was performed according to the principles of the Declaration of Helsinki, and consent was obtained from each subject after full explanation. In family 1 -80% of the subjects agreed to take put,
Family5
I
8
8
in the other families all participated in the study. For each subject, we used a structured questionnaire for lifestyle factors such as smoking and alcohol intake. In particular, we investigated the use of antisecretory therapy, non-steroidal anti-inflammatory drugs (NSAIDs) and antimicrobial drugs over the previous month. Subjects using antisecretory therapy, NSAIDs and antimicrobial drugs during the last month were excluded from the study to allow the necessary washout period for drug assumption. We also excluded those patients, in Family 1, that have been treated with eradication therapy for H. pylori infection. Blood samples were taken at the time of the medical interview and stored for linkage analysis and to determine serum PGA and PGC levels, gastrin levels, H. pylori antibodies, hepatitis A (HAV) antibodies and blood groups. The method available to localize the gene of the disease is linkage analysis of the cosegregation. Linkage analysis can also be applied when penetrance in unknown and can also be extended to situations in which two or more genes play a role in the inheritance of a disease 28. We have examined the segregation of PGA and PGC with microsatellite dinucleotide repeat DNA markers along chromosome 11 and 6. Since PG genes are localized to the centromere of the human chromosome 11 in the region p I 1+q13 I5 I7 and in the region p2 1.1 of human chromosome 6 Ix, we studied the cosegregation with ulcer disease analyzing the border on markers. These markers were Dl lS480 and PYGM for chromosome 11, D6S 105, D6S1610 and TRMI for chromosome 6. Genomic DNA was extracted according to the salting out procedure. DNA samples were analysed by polymerase chain reactions (PCR). The PCR were performed as follows: 50 ng of genomic DNA was used as template with 10 pM each oligonucleotide primer, 0.4 units of Taq polymerase, 67 mM Tris-HCl (pH 8.8), 16.6 mM ammonium sulphate, 1.5 mM MgCl 2, 0.01% Tween 20, and dGTP, dATP, dTTP, and dCTP each at 100 pM in a final reaction volume of 12 pl. The samples were over-layed with 15 pl of mineral oil to prevent evaporation. PCRs were performed in an MJ Research microplate thermocycler under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The cycle was repeated 30 times. After PCRs, 2~1 aliquots of the reaction mixture were denatured and separated on a 9% denaturing polyacrylamide gel. Gels were then silver stained and dried. Serum PGA and PGC concentrations were determined by PGA and PGC RIA kit (Sorin Biomedica, Saluggia, Italy); the limits of detection were 30-100 pg/l and 2-14 pg/l for PGA and PGC, respectively. The serum gastrin levels were also determined by a
10 11 13 12
Fi3.3. Family 5 pedigree, living near the Val di Resia, with history of peptic disease. For explanation: see legend Figure I.
T. Del Bianco et al.
RIA kit to (Sorin Biomedica, Saluggia, Italy); the normal range established being 0- 115 rig/l. The presence of anti-H. pylori IgG antibodies was determined by an in-house, enzyme linked immunoassay (RADIM, Pomezia, Italy); a threshold titre >20 U/l was used to discriminate H. pylori positive from H. pylori negative individuals. Sera were tested also by ELISA for anti-HAV (Abbott, Campoverde, Italy); the results were expressed in positive or negative for HAV infection. ABO blood groups were determined by a standard haemagglutination assay performed with a 3% suspension of peripheral blood. Linkage analysis was performed using a computer programme named LINKAGE (version 5.1) which estimates the lod-score [an acronym for logarithm of the log odds ratio (OR)], which is the ratio
Fmnlly 2
log10 = LP) L; J--l~ where L(8) is the likelihood function of the samp,c and 8 is the maximum likelihood estimator of the recombination fraction. In practice, lod-scores are evaluated at a limited number of points OS&‘/Z and the value that maximises the lod-score is chosen as the estimate of 19.A lod-score was calculated by assuming 0.7 and 0.9 penetrance. A lod-score of 3 or greater is usually taken as significant evidence of linkage; a lod-score of -2 or lower is taken as evidence of no-association 2y. The association between H. pylori infection and blood group, HAV infection, peptic disease and serum PGC levels was analysed separately using log linear models and OR was used as a measure of the association together with 95% confidence intervals (95% CI) 30.
Results
The serum PGA values show different levels in selected families with peptic disease (Fig. 4). We have studied the serum PGA values and the presence of elevated titres of H. pylori antibodies. If there were high PGA and high H. pylori antibodies, the value of PGA was considered associated with H. pylori infection. We, therefore, began performing linkage analysis in Family 1 because these subjects were found to have peptic disease with high levels of PGA. Furthermore in each family member, serum gastrin levels were normal (data not shown). According to epidemiological studies in the Family considered, there is a
Fig. 4. Serum PGA and anti-H. py/ori levels in families 1, 2 and 5 (see pedigree for references to subjects).
progressive increase in the prevalence of H. pylori infection with age. Even if there is no close correlation between serum PGA levels and serum Anti H. pylori levels, this family is particularly indicated for linkage study because there are several affected individuals who could have inherited the same disease-causing allele from a common ancestor. It is a genetically isolated population. It is possible to exploit information from many historical meioses and, thereby, afford much higher recombinational resolution. In fact, in this analysis, the classical strategy is, first, to detect a major-gene effect by segregation analysis and, second, to seek for linkage with genetic markers by the lod-score method using major-gene parameters. The localisation of the human PGA gene cluster is to the chromosome 11 and, for linkage study, two DNA markers from this region were used. The maximum 15
Peptic ulcer inheritance
kbk I. Lod-score test values at each 0 level for different markers used.
lod score values were -0.049 at eO.4 (70% penetrance) and -0.06 at 0=0.4 (90% penetrance) for Dl lS480 DNA marker, and 0.017 at 60.4 (70% penetrance) and 0.031 at eO.4 (90% penetrance) for PYGM DNA marker. In every examined marker the lod-score test was ~3, thus linkage analysis provided no evidence for significant causal mutations of PGA genes (Table I). The lod-score is not equal or lower than -2 so that linkage analysis cannot accept any association. Of the 20 patients with peptic ulcer disease, eight subjects had never used NSAIDs and four subjects (1114,1115,1116,11123) do not have H. pylori infection. In order to gain a better insight into the genotype risk, the odds of the presence of a small fragment (4.6) (Fig. 5) associated with no H. pylori infection was estimated: 9=
P (fragment
= (4*6)/H.
pylori
= no)
P fragment
*(4-6)/H.
pylori
= no)
We find Y=3, so the probability of having peptic disease for subjects with the same small fragment, without H. pylori infection, is three time greater than the probability of falling ill for a patient without that fragment (Table II). This value, of course, does not support any statistical conclusion because of the very small sample size, however, data show strong evidence of an important effect of the genetic component
4. I. 6meB fragment 46 of chromoeome 11 in ragion ql I-pter. DNA samples were anelysed by poIymerase chain &on. In lane 3, 5. 6 and 8, indicated by arrows, the ekctfwhoretic patterns are identical for the markers amplified from the pwted DNA semples. The fragment 4.6 indicates the haplotype of the disease.
16
I
NW II. Contingency table of fwquertcies between H. p@ri infection and haplotype.
4.6 e4.6
i
3 1
:
4
4
8
on presence of the disease. Moreover, these four patients had elevated values of serum PGA levels. The linkage analysis was performed also for DNA markers along chromosome 6; the results obtained neither exclude nor confirm the association. We also observed that these patients do not show a uniform genetic profile. At this stage of our work, we did not perform the linkage analysis in members of the other families. We thought it better to first find a localisation in the family potentially more informative for disease and then, in other families to test, only the markers near that localised gene. To emphasize our genetic results, we made some statistical considerations on other variables usually considered in the literature. We analyzed the association between H. pylori infection, blood groups, HAV infection and serum PGC levels in Family 1 and in other family members with a history or no history for peptic disease living in this valley and in a nearby valley. We studied a total of 78 subjects. Seropositivity for H. pylori infection is not associated with peptic ulcer disease either Family 1 (p=O.8) or in other families members (p=O.2) even if there is more evidence of independence in data of Family 1. The association between H. pylori infection and blood group 0 is not statistically significant (p=O.504 Family 1 and p=O.533 other families). Furthermore, there is no association between PGC and H. pylori infection (p=O.5 Family 1 and p=O.O6 in other families). As far as concerns the relation between HAV
We III. Analysis of association between H. py/ori infection, blood groups, HAV infection, peptic disease and PGC serum levels in Family 1 and other families members (with peptic disease and control families. Total number of subjects: 781.
LIIT
lbwrlrpir Blood GMUPS vs H. pybri infection HAV infection vs H. pybri infection Peptic disease vs tl. &ori infection PGC vs H. py/wi infection
Family 1 Other families Family 1 Other families Family 1 Other families Family 1 Other families
and H. pylori infection, log-linear analysis shows different results for the two groups: in Family 1 there is independence (p=O.527) while in the other family subjects there would appears to be an association (p=O.Ol; OR=2.208, 95% CI 1.16 to 4.21) (Table III). Discussion In this study, we have identified a family with a high prevalence of peptic ulcer and with high serum PGA levels. Moreover, there is a particular environmental situation. In fact, members living in a valley of Northeast Italy, belong to an ancient barbaric nation, the Ruteni, who, remained isolated, for a long time, from neighbouring communities. Mountains enclose this valley so that, until recent times, there was only one route of access at the bottom of these mountains. The Resiani comprise the most part of the population, only 2.9% of the community members come from other areas. There is a tendency to marry within the same settlement 3’-33. Because of these characteristics, Resiani could, indeed, have formed a rarely genetic isolated found, so they are ideal for genetic studies. The four affected members with no important risk factors show the same fragment of gene localised in the same region to chromosome 11. When in a linkage analysis between a disease and a genetic marker some families show linkage while others do not, then the question often asked is whether the observed variation in the recombination fraction over families is due to change alone or whether it might be due to disease heterogeneity. Linkage is due to the phenomenon that alleles at different loci on a single chromosome are often transmitted together from parent to offspring. On the other hand, even when the loci are physically close, alleles on different homologous chromosomes are sometimes transmitted to one offspring. When the
0.44 0.38 0.39 6.52 0.06 1.49 0.44 3.56
P
OR
i:g 0.52 0.01
2.208
95%lFl
1 .I6421
0.80 0.20 0.50 0.06
latter occurs, a recombination event is said to have separated the two loci 34. In our family, the meiotic events analysed were low because only four subjects have the fragment 4.6, and this situation is not particularly favourable. Since phenotypes are function of the genotypes (our subjects had high serum PGA levels), the probability of the observed phenotypes should be compatible with genotypes 35. Our analysis provided no evidence for significant causal mutations within the region bounded by Dl lS480 and PYGM on chromosome 11. But genetic analysis of complex traits is complicated by many factors: reduced penetrance, aetiologic heterogeneity, the number of loci contributing to disease (and their interactions), mode of inheritance, presence of phenocopies (non-genetic cases), etc. It is still possible, however, that a gene of major effect exists in this region or in another region or in another chromosome, either with low penetrance or with heterogeneity. We think genetic heterogeneity is more likely because the other family members showed no linkage with any of the investigated loci. Some diseases were previously mapped to a chromosome, and other studies had demonstrated that the same disease appears to be transmitted with polymorphic DNA markers by another chromosome 36. Another point is that for most of the models, the use of major gene parameters in lod-score analysis leads to a large bias in estimation of the recombination fraction and sometimes also to a rejection of linkage for the true combination fraction 37. Genetic factors alone, without environmental interactions, could account for this segregation, and vice versa if environmental interactions are important, our sample is ideal because there are subjects with hyper PGA and peptic ulcer and without hyper PGA and peptic ulcer, living in and outside the valley. This study supports the idea of the peptic ulcer inheritance at least in a small group of patients without the common factors of risk such as H. pylori infection and NSAIDs use. So, we are, therefore, encouraged to perform
Peptic ulcer inheritance
linkage analysis in the entire DNA, in our entire sample, to demonstrate or to exclude the heredity. Our hypothesis is also supported by the fact that in Family 1 the data lead to conclusions different from those in the literature. -H. pylori is a very common infection and causes peptic ulcer. In Family 1 H. pylori infection and peptic disease are independent. - Both PGA and PGC are present in the chief cells of the oxyntic glands of the gastric corpus mucosa, but only PGC is present in the gastric antrum. Colonisation of the gastric mucosa by H. pylori is associated with histologically confirmed chronic gastritis. Increased serum PGC concentrations appear to be related to the severity of gastritis in H. pylori infected subjects 38 39. Since peptic ulcer is associated with H. pylori infection, we should find a link also in Family 1 between disease and serum PGC. But as ulcer disease is independent from H. pylori infection, as it is independent from serum PGC levels. - Boren et al. 4” demonstrated that the receptor for H. pylori on the gastric epithelial cell is the blood group antigen Lewis b. Bacteria did not bind to Lewis b antigen substituted with a terminal GalNAcal-3 or Gala1-3 residue (blood group A and B determinants), suggesting that the availability of H. pylori receptors might be reduced in individuals with blood groups A and B compared with those with blood group 0. This speculation seemed to be consistent with the old observation that peptic ulcer is more prevalent in individuals with blood group 0 4’ 42. Recently it was demonstrated that H. pylori adheres to isolated human gastric cells is a manner that is not dependent on Lewis antigen expression on these cells, and expression of Lewis antigens on the surface of gastric cells is not dependent on Lewis antigen expression on erythrocytes 43. In our data there is no evidence of this relationship. - If person-to-person contact plays an important role in H. pylori transmission, the infection seems very similar to that of hepatitis A virus. Some Authors have shown that the seroprevalence of H. pylori infection parallels HAV, suggesting similar modes of transmission 44, others have shown no statistically significant correlation between seroprevalence of H. pylori and HAV 4546. In our results, the relationship between seroprevalences of H. pylori and HAV was not statistically significant in Family 1 whereas it was in the other families. Thus, this study underlines the possible role of the peptic ulcer inheritance (investigated by means of the serum PGA distribution) in patients with a low rate of H. pylori infection located in a rare genetic and environmental isolated situation. 18
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