Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis

Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion -28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

Purine nucleoside phosphorylase deficiency: a new case report and identification of two novel mutations (Gly156A1a and Val217Ile), only one of which (Gly156A1a) is deleterious

Purine nucleoside phosphorylase (PNP) deficiency results in an autosomal recessive immunodeficiency disease characterized by initial involvement of cellular immunity and neurological manifestations with subsequent abnormalities of humoral immunity. The initial presentation and clinical course has varied widely in the relatively few published cases. The molecular basis has been reported in only 10 patients, precluding evaluation of phenotype-genotype relationships. We now report clinical, immunologic, and molecular findings in a new case of relatively early onset that emphasizes hypotonia and developmental delay as early manifestations. The patient carried two novel missense mutations (Gly56A1a and Val217Ile) on the same allele in apparent homozygosity. Expression of each of the mutant enzymes in vitro demonstrated that the Gly156A1a mutation abolished enzyme activity while the Val217Ile mutation was without obvious effect and is therefore a normal variant. Such "normal" polymorphisms might be associated with a variable response to the immunosuppressive PNP inhibitors currently in clinical trials.     

Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies

The Marfan syndrome (MFS) is a pleiotropic, autosomal dominant disorder of connective tissue with highly variable clinical manifestations including aortic dilatation and dissection, ectopia lentis, and a series of skeletal anomalies. Mutations in the gene for fibrillin-1 (FBN1) cause MFS, and at least 337 mainly unique mutations have been published to date. FBN1 mutations have been found not only in MFS but also in a range of connective tissue disorders collectively termed fibrillinopathies ranging from mild phenotypes, such as isolated ectopia lentis, to severe disorders including neonatal MFS, which generally leads to death within the first two years of life. The present article intends to provide an overview of mutations found in MFS and related disorders and to discuss potential genotype-phenotype correlations in MFS.     

一个遗传性痉挛性截瘫家系的临床特点与spastin基因突变分析

目的探讨遗传性痉挛性截瘫(hereditary spastic paraplegia,HSP)一家系的临床特点及其与spastin基因突变关系。方法对整个家系进行详细的临床检查,先证者和家系内另两例患者进行了肌电图检查,先证者还进行了胸髓核磁共振检查。应用聚合酶链反应结合DNA序列分析方法,检测该家系中先证者和其父亲spastin基因的突变情况。结果家族中所有患者具有遗传性痉挛性截瘫的典型表现,先证者胸髓核磁共振成像显示胸髓明显萎缩,PCR-DNA序列分析患者spastin基因的17个外显子均未发现有异常的突变。结论该HSP家系的患者具有典型的临床表现,并非spastin基因外显子突变所致。     

V180I mutation of the prion protein gene associated with atypical PrP glycosylation

A valine to isoleucine mutation at residue 180 was identified in a French patient with Creutzfeldt-Jakob disease (CJD). The mutation is located in the close vicinity of one of the two N-glycosylation sites of the cellular prion protein (PrP^C). Western blot analysis revealed accumulation in the brain of the pathogenic proteinase K-resistant PrP (PrP^Sc) isoform with the notable absence of the diglycosylated band. The mutant protein expressed in CHO cells was correctly glycosylated, suggesting that the atypical glycosylation pattern of PrP^Sc was not due to the mutation at position 180. These results suggest that the diglycosylated form of the mutant PrP^180I prevents its conversion into the pathogenic mutant form PrP^Sc180I, supporting a central role of N-linked glycan chains in the PrP conversion process.     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Three novel activating mutations in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia

We report three novel activating mutations in the calcium-sensing receptor (CASR) that are responsible for autosomal dominant hypocalcemia (ADH) in three unrelated families. Each mutation involves a missense substitution resulting in a nonconservative amino acid alteration, P221L, E228Q, and Q245R. These mutations were observed in affected family members, but not in unaffected family members or in unrelated control samples. All three mutations are clustered in the extracellular domain of the CASR in a region dominated by negatively charged amino acids. Each mutant and wild-type receptor was expressed in Cos-1 cells. A luciferase reporter gene assay was utilized to detect the level of receptor activity by utilizing a protein kinase C-activated promoter to drive the production of luciferin, the reporter gene product. All three mutant receptors exhibited an increased sensitivity to calcium at all concentrations tested when compared to the wild-type receptor, supporting the hypothesis that these are activating mutations and are responsible for the ADH phenotype in these families. The data presented in this study suggest the importance of this highly negatively charged region of the extracellular domain in normal CASR function.