R117H Mutation Research Articles

Chronische Pankreatitis – Pankreaskarzinom: Einfluss genetischer Faktoren

Chronic pancreatitis: Only recently mutations in several genes were found in patients with chronic pancreatitis. In those with a familial chronic pancreatitis mutations of the cationic trypsinogen were identified and the variants N29I and R122H lead to an autosomal dominant disease. In this group of patients the mutation N34S of the trypsin inhibitor SPINK1 was detected. In so-called idiopathic pancreatitis both variants of the SPINK1 and of the CFTR (cystic fibrosis transmembrane conductance regular) were identified. Alterations in both genes were also found in patients with alcoholic chronic pancreatitis. The strongest risk factor for chronic pancreatitis were trypsinogen mutations N29I and R122H mutations. However, both SPINK1 and CFTR increased the risk for chronic pancreatitis to a higher level than alcohol consumption. A genetic investigation should be performed in familial disease and younger age, but also in patients without family history and higher age a mutation could be found. Pancreas cancer: In 10% of the patients with pancreas cancer other members of the family were affected from the disease. Some of them belong to well characterized familial syndroms like HNPCC or Peutz-Jeghers-syndrom. In a minority of the others a genetic factor may be found, too. In sporadic disease the development of the tumor is characterized by continued acquirement of genetic alterations described by the PanIN model (pancreatic intraepithelial neoplesia). This means that the evolution of the neoplasia progresses from normal tissue via epithelial hyperplasy (PanIN 1A), papillary hyperplasy without (PanIN 1B) and with dysplasy (PanIN 2) and carcinoma in situ (PanIN 3) to invasive pancreas cancer. The progression is associated with genetic alterations of the cells (mutations of ki-ras, p16, p53 etc.). This results in deterioration of control of the cell cycle and the apoptosis and explains the malignancy of the disease. These findings may be used in the future to develop newer therapeutic principles in order to improve the dismal prognosis of this disease.

Cystic fibrosis genotype and bacterial infection: a possible connection

Patients with cystic fibrosis (CF) have repeated bacterial infection of the airways, which can lead to chronic infection. There is evidence that disease severity is determined by the genetic mutations present. This study aims to establish if CF genotype is related to the frequency and types of airway bacterial infection. Adult patients attending the regional CF unit are followed for two years and assigned to one of three groups depending on whether they are chronically infected with Burkholderia cepacia complex (BCC) organisms, Pseudomonas aeruginosa, or neither of these organisms. Genotype analysis is performed on all patients to determine which of the cystic fibrosis transmembrane regulator (CFTR) gene mutations are present. The numbers and types of organism with the CFTR mutations isolated from sputum are identified. Data are available on 59 patients: 15 colonised with BCC, 24 colonised with P. aeruginosa, and 20 not colonised with either organism. Twenty patients were homozygous for ∆F508, 25 were heterozygous, and the ∆F508 mutation was not present in the remaining 14 patients. Patients homozygous or heterozygous for the ∆F508 mutation had an increased likelihood of colonisation with BCC or P. aeruginosa, an increased number of positive sputum cultures and a higher frequency of multiple infecting organisms. Cystic fibrosis mutational analysis identified seven patients who had the R117H mutation. These patients were less likely to be colonised with BCC or P. aeruginosa. In conclusion, patients homozygous or heterozygous for the ∆F508 deletion are more likely to suffer airway colonisation with BCC or P. aeruginosa, with increased numbers of positive sputum cultures and infecting organisms. Those with the R117H mutation are less likely to be colonised by Gram-negative organisms.

First Study of CF Mutations in the CFTR Gene of Iranian Patients: Detection of F508, G542X, W1282X, A120T, R117H, and R347H Mutations

Thirty-seven unrelated Iranian CF families were screened for the presence of seven common mutations (DeltaF508, G542X, W1282X, G551D, N1303K, 1717-1G-->A, and 621-1G-->T) using ARMS PCR and exons 4 and 7 of the CFTR gene by SSCP method. This study resulted in the identification of 26.8 per cent of all CF alleles: DeltaF508 (16.2 per cent), W1282X (4 per cent), G542X (2.7 per cent), R117H (1.3 per cent), R347H (1.3 per cent), and A120T (1.3 per cent) mutations were detected. To the best of our knowledge, it is the first report of an Asian subject carrying the A120T mutation. Our findings suggest heterogeneity in the Iranian population, stressing the need to draw attention to sequence analysis in order to find population-specific mutations.

Etiology of azoospermia in 100 consecutive nonvasectomized men

History was taken systematically for 100 azoospermic, nonvasectomized men referred consecutively to a Danish fertility clinic. The men were examined by ultrasound, and their blood samples were analyzed for karyotype, Y microdeletions, and cystic fibrosis transmembrane conductance regulator gene mutations. In 29% of patients, the condition could be explained by genetic abnormalities; in 22%, by diseases or external influence; and in 27%, by former cryptorchidism. The azoospermic condition remained unexplained in only 22%.

Microsphere Bead Arrays and Sequence Validation of 5/7/9T Genotypes for Multiplex Screening of Cystic Fibrosis Polymorphisms

The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

Hereditary pancreatitis: clinical characteristics and diagnostic criteria in Japan.

Hereditary pancreatitis (HP) is the strongest known risk factor for pancreatic cancer. The aim of the present study is to establish diagnostic criteria for HP to predict and identify high-risk groups for pancreatic cancer. We collected clinical data for 210 patients with recurrent acute or chronic pancreatitis, and examined mutations of the cationic trypsinogen (CT) gene in 57 patients with a family history of pancreatitis or with early-onset idiopathic recurrent acute or chronic pancreatitis (40 years of age or younger). DNA was extracted from peripheral blood leukocytes, and exons 2 and 3 of the CT gene were individually amplified by polymerase chain reaction (PCR) and sequenced. Of these 57 patients in whom mutations of the CT gene were examined, the R122H (20 patients) and N29I (5 patients) mutations in the CT gene were observed in 25 patients (43.9%). From the analysis of clinical records and the CT gene of these patients, we proposed the following adaptations to the diagnostic criteria for HP: (1) at least one of the affected members in a family has no known etiological factors, (2) we deleted the definition of "different generation", but included the upper limit of the age of onset of pancreatitis in the case of siblings (at least 1 of the patients in a family <40 years of age). According to these criteria, all patients with the CT gene mutations in the present study could be classified as having HP, with the exception of 2 sporadic cases with the R122H and N29I mutations, respectively. Based on these findings, we revised the criteria for the diagnosis of HP; (1) recurrent acute or chronic pancreatitis with R122H or N29I mutation of the CT gene, or (2) recurrent acute or chronic pancreatitis with a family history of 2 or more affected patients, irrespective of generation, with at least 1 of the patients having no known etiological factors, and in case of siblings only, the onset of the disease in at least 1 of the patients is under age 40 years. The revised criteria in the present study are appropriate and of clinical usefulness to diagnose patients with HP even in cases without the genetic testing. However, if and when more genes are detected, it will be important to reexamine the mutation-negative patients now classified as HP based on our proposed criteria.

The genetics of Shwachman-Diamond syndrome

The genetics of Shwachman-Diamond syndrome

Mutations of the cationic trypsinogen gene in patients with hereditary pancreatitis.

Hereditary pancreatitis has been shown to be caused by one of two mutations (R117H and N21I) of the cationic trypsinogen gene (PRSS1). Families with hereditary pancreatitis in the north of England were investigated for these mutations. The clinical features associated with each mutation were compared. In individuals from nine families with hereditary pancreatitis, DNA was screened for the R117H and N21I mutations. All five exons of the cationic trypsinogen gene were also sequenced to search for additional mutations. Haplotype analysis was carried out to identify common ancestors. Clinical data were collected. The R117H mutation was identified in three families and N21I in a further five. The R117H mutation was associated with a more severe phenotype than N21I in terms of mean(s.d.) age of onset of symptoms (8.4(7.2) versus 16. 5(7.1) years; P = 0.007) and requirement for surgical intervention (eight of 12 versus four of 17 patients respectively; P = 0.029). Haplotype analysis suggested that each mutation had arisen more than once. Two mutations in the cationic trypsinogen gene cause hereditary pancreatitis in eight of nine families originating in this region. The R117H mutation is associated with a more severe form of the disease in terms of age at onset of symptoms and requirement for surgical intervention.

Mutational screening of patients with nonalcoholic chronic pancreatitis: identification of further trypsinogen variants

OBJECTIVES: Mutations of the cationic trypsinogen (CT) and the serine protease inhibitor, Kazal type 1 (SPINK 1) are associated with chronic pancreatitis. After mutational screening of a cohort of patients with nonalcoholic chronic pancreatitis, we report three novel variants of the trypsinogen molecule and the clinical characteristics of the carriers. METHODS: The coding region of the exon 2 and 3 of the CT gene of 523 patients with chronic nonalcoholic pancreatitis (108 patients with suspected hereditary pancreatitis (HP) and 415 patients with “idiopathic” pancreatitis [IP]) and 82 controls was analyzed after polymerase chain reaction amplification. Clinical characteristics were obtained by questioning the patients and their relatives and physicians. HP was suspected when two members of a family had chronic pancreatitis. A restriction digestion was used to analyze the N34S mutation SPINK 1. RESULTS: The mutation R122H of the cationic trypsinogen was found in 21 index patients, N29I in six index patients, and A16V and D22G in one index patient, all from HP families. The N34S mutation of SPINK 1 was found in two index patients with a family history of HP. In three patients, the novel point mutations L104P, R116C, and C139F of the cationic trypsinogen were found. A clear autosomally dominant inheritance of chronic pancreatitis was not present in these families. In 75 index patients from HP families (69.4%), no mutation could be found. The SPINK 1-mutation N34S was detected in only one patient carrying a CT mutation, and was found in 68 (16.4%) of patients with IP. CONCLUSIONS: The R122H and N29I mutations of CT are the most common disease-associated mutations in HP; the N34S mutation of SPINK 1 is the most frequent genetic risk factor associated with IP. The CT gene carries several variations that could be associated with chronic pancreatitis. To avoid overestimating the pathogenetic impact of novel trypsinogen variants, a detailed clinical characterization of all patients with early onset chronic pancreatitis is mandatory.

Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C.

Compound heterozygotes for a severe cystic fibrosis transmembrane conductance regulator (CFTR) mutation and the R117H or R117C mutation (R117H/C) have clinical presentations that vary from classic cystic fibrosis (CF) to an incidental genetic finding. The aim of this study was to assess the influence of the intron-8 polythvmidine sequence (IVS8) on the relationship between genotype and phenotype of individuals with R117H/C. All individuals with R117H/C known to CF clinics in Australia and New Zealand were retrospectively studied by collecting information on genotype, age, pancreatic status, sweat electrolytes, sputum microbiology and pulmonary function. Forty-one individuals (39 with R117H and two with R117C), 16 on an IVS8-5T background and 25 on an IVS8-7T background were identified. Twelve individuals presented clinically, four were siblings of known R117H/C compound heterozygotes and 25 were detected by newborn screening. Eleven of 14 of the IVS8-5T group (78%) with sweat chloride results available had sweat CI > 60 mmol x L(-1) compared to 5 (20%) of the R117H/7T group (Chi-squared=10.4, p=0.001). Two were pancreatic insufficient, both IVS8-5T. Two IVS8-5T individuals have recently died (aged 43 and 19) and of the 14 surviving IVS8-5T group, 11 (79%) are symptomatic compared to eight (32%) of the IVS8-7T individuals (Chi-squared=6.1, p=0.01). In conclusion, most individuals with R117H/C on a IVS8-5T background have an elevated sweat chloride and clinical cystic fibrosis, which in some cases is severe. Most individuals with R117H/C on an IVS8-7T background do not have clinical cystic fibrosis but should be followed for the development of clinical disease.

A new polymorphism for the RI22H mutation in hereditary pancreatitis

BACKGROUND AND AIMSHereditary pancreatitis (HP) is a rare form of recurrent acute and chronic pancreatitis. Mutations in the cationic trypsinogen (protease serine 1, PRSS1) gene have been identified as causing...

Mutations of the Cationic Trypsinogen Gene in Hereditary and Non-Hereditary Pancreatitis

Background/Aims: Mutations in the cationic trypsinogen gene have been detected in patients with hereditary pancreatitis (HP). This study investigates the prevalence of the R122H, N29I, A16V and –28delTCC mutations in the common, non-hereditary forms of chronic pancreatitis and in a HP family. Methods: DNA was prepared from blood samples of 53 patients with chronic pancreatitis (36 alcoholic, 14 idiopathic and 3 hereditary), 20 alcoholic controls and 20 healthy, ethnically matched controls. The R122H and A16V mutations were identified by the polymerase chain reaction (PCR) and restriction enzyme digestion. A nested-PCR was used to identify the N29I mutation. The –28delTCC deletion and the C133807T polymorphism were sought by direct sequencing. Results: The R122H mutation was detected in 1 patient with alcoholic chronic pancreatitis and all 3 affected members of a HP family. The N29I, A16V and –28delTCC mutations were not detected in any of the study subjects. At the C133807T polymorphism, the C allele and C/C genotype were significantly increased in alcoholic chronic pancreatitis (p = 0.001 and p = 0.0004, respectively) while the T allele and CT genotype were significantly reduced (p = 0.001 and p = 0.004, respectively) compared to healthy controls. Conclusions: Mutations of the cationic trypsinogen gene are rarely found in chronic pancreatitis patients of typical aetiology. Screening for these mutations should be considered in those with a family history consistent with hereditary pancreatitis but may also be appropriate in a well-defined subgroup of patients with non-hereditary chronic pancreatitis, i.e. those who have developed the disease before the age of 30.

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N211, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

Molecular screening of the CFTR gene in men with anomalies of the vas deferens: identification of three novel mutations.

Many studies have shown that congenital absence of the vas deferens (CAVD) is a genital cystic fibrosis transmembrane conductance regulator (CFTR)-mediated phenotype, with a broad spectrum of abnormalities causing male infertility. The genotype of these patients includes mutations in the CFTR gene, e.g. DeltaDeltaF508, R117H and the T5 allele; all of which are commonly found in CAVD. In this study we have screened the entirety of CFTR gene in 47 males with anomalies of the vas deferens: 37 cases of congenital bilateral absence of the vas deferens, three cases of congenital unilateral absence of the vas deferens and seven cases of obstructive azoospermia with hypoplastic vas deferens. Among the 94 chromosomes studied, 65 mutations, of which three are novel (2789+2insA, L1227S, 4428insGA), were identified. The majority of patients (63.8%) had two detectable CFTR gene mutations. Furthermore, high frequencies of the DeltaDeltaF508 mutation (44.7%), the T5 allele (36.2%) and R117H mutation (19.1%) were observed.

Gene Conversion-like Missense Mutations in the Human Cationic Trypsinogen Gene and Insights into the Molecular Evolution of the Human Trypsinogen Family

Over the past decade, gene conversion has been shown increasingly to be a cause of human disease. Through this process, a functional gene is converted into a mutant by a homologous, nonfunctional one. In this article, we demonstrate that gene conversion is a likely cause of the mutations of the human cationic trypsinogen (PRSS1) gene that are associated with hereditary or sporadic pancreatitis, including the R122H (CGC>CAT: c.365–366 GC>AT), N29I (AAC>ATC: c.86A>T), and A16V (GCC>GTC: c.47C>T) missense mutations. This hypothesis is strongly supported by four lines of observation. First, human group I trypsinogen genes are tandemly repeated and share a high sequence homology between them. Secondly, a possible donor sequence for each variant is present in the PRSS1 gene's paralog(s). Thirdly, there exist uninterrupted sequence tracts ranging from 30 to 114 bp in the putatively converted regions. Finally, Chi-like and palindromic sequences are found in the vicinity of these missense mutations. This theory, if correct, will make the pancreatitis-associated PRSS1 mutations a unique example, as it shows that a functional gene may be converted by several paralogs, and that such an event may even occur between two functional genes (i.e., the N29I mutation), resulting in disease. This adds further to the diversity of genetic mechanisms underlying human disease. In addition, this genetic finding provides, for the first time, concrete evidence of the contribution made by gene conversion to the molecular evolution of the human trypsinogen family.

This month in J Lab Clin Med: Issue highlights for October 2000

This month in J Lab Clin Med: Issue highlights for October 2000

R117H and IVS8-5T Cystic Fibrosis Mutation Detection by Restriction Enzyme Digestion

R117H and IVS8-5T Cystic Fibrosis Mutation Detection by Restriction Enzyme Digestion

Clinical Features Associated with a Delayed Diagnosis of Cystic Fibrosis

Background: The majority of patients with cystic fibrosis (CF) are diagnosed in the first decade of life. In a small number of patients, the diagnosis is not made until later. Objective: In this study, the clinical and genetic features of patients diagnosed after the age of 10 were examined. Methods: All living patients in Northern Ireland diagnosed prior to 1983, when neonatal screening was introduced, were studied. A total of 103 patients were identified of whom 18 were diagnosed after the age of 10. The relationships between late diagnosis and clinical presentation, sputum microbiology, pancreatic sufficiency, nutritional status, genotype and distance from the regional CF centres was determined by multiple regression analysis. Results: All 18 late-diagnosed patients had a sweat (chloride >70 mmol/l). Late diagnosis was significantly related to carriage of the R117H mutation (r² = 0.45) and pancreatic sufficiency (r² = 0.37). There was a weak relationship with pulmonary function (r² = 0.09). Conclusions: In Northern Ireland, late diagnosis in mainly associated with pancreatic function and carriage of the R117H mutation.

Evidence that hereditary pancreatitis is genetically heterogeneous disorder

Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute attacks of severe abdominal pain with an onset in early childhood. Many HP patients progress to complicated chronic pancreatitis and/or pancreatic cancer. Initially, a single mutation R117H in the cationic trypsinogen gene was detected in all affected members of five unrelated HP families. Further studies identified a second mutation (N21L) in two HP families without the R117H mutation. Before the association between cationic trypsinogen and HP was found, we detected a cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation (L327R) in all affected individuals of a family with HP. We therefore performed a mutational analysis for R117H and N21L in cationic trypsinogen in this and three additional unrelated families with HP. The R117H mutation was detected in all 9 affected members of three HP families and in 3 asymptomatic but at-risk relatives. However, neither the R117H nor the N21L mutation in the cationic trypsinogen were found in the HP family with the L327R alteration in CFTR. The L327R allele segregates with the disease within this HP family and was not detected on 360 unrelated Caucasian non-CF chromosomes. Although close to 800 different mutations have been detected in the CF gene of cystic fibrosis patients, L327R is a new alteration, not yet reported in connection with CF. The results of this study indicate that the CFTR gene may play a role in the etiology of minority of cases with HP and suggest that hereditary pancreatitis is genetically heterogeneous disease.

Evidence that hereditary pancreatitis is genetically heterogeneous disorder.

Hereditary pancreatitis (HP) is an autosomal dominant disorder characterized by recurrent acute attacks of severe abdominal pain with an onset in early childhood. Many HP patients progress to complicated chronic pancreatitis and/or pancreatic cancer. Initially, a single mutation R117H in the cationic trypsinogen gene was detected in all affected members of five unrelated HP families. Further studies identified a second mutation (N21L) in two HP families without the R117H mutation. Before the association between cationic trypsinogen and HP was found, we detected a cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation (L327R) in all affected individuals of a family with HP. We therefore performed a mutational analysis for R117H and N21L in cationic trypsinogen in this and three additional unrelated families with HP. The R117H mutation was detected in all 9 affected members of three HP families and in 3 asymptomatic but at-risk relatives. However, neither the R117H nor the N21L mutation in the cationic trypsinogen were found in the HP family with the L327R alteration in CFTR. The L327R allele segregates with the disease within this HP family and was not detected on 360 unrelated Caucasian non-CF chromosomes. Although close to 800 different mutations have been detected in the CF gene of cystic fibrosis patients, L327R is a new alteration, not yet reported in connection with CF. The results of this study indicate that the CFTR gene may play a role in the etiology of minority of cases with HP and suggest that hereditary pancreatitis is genetically heterogeneous disease.