Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography

BACKGROUND: R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365~366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. MATERIALS AND METHODS: DNA samples containing either the known c.365G>A or c.365~366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay. RESULTS: DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C). CONCLUSIONS: A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation

Detection of two Alu insertions in the CFTR gene

Background LINE-1 (long interspersed element-1) or L1-mediated retrotransposition is a potent force in human genome evolution and an occasional cause of human genetic disease. Since the first report of two de novo L1 insertions in the F8 gene causing hemophilia A, more than 50 L1-mediated retrotranspositional events have been identified as causing human genetic disease. However, a significant bias has generally militated against the detection of these pathological events at autosomal loci. Based upon this and other observations, we surmised that some previously unresolved cystic fibrosis chromosomes might carry hitherto undetected L1-mediated retrotranspositional insertions at the CFTR locus. This study represents an attempt to identify such mutational events. Methods 100 previously unresolved cystic fibrosis chromosomes were carefully reanalyzed using quantitative high-performance liquid chromatography (QHPLC). Results Two simple Alu insertions were identified in the CFTR gene, within exons 16 and 17b respectively. Conclusions Our findings have not only revealed a previously unknown mutational mechanism responsible for cystic fibrosis but also represent an important addition to the already diverse spectrum of known CFTR gene mutations. Experience with the CFTR gene suggests that pathological L1-mediated retrotranspositional events may also have been overlooked at other gene loci and should always be considered in cases that appear to be refractory to analysis

The hereditary pancreatitis gene maps to long arm of chromosome 7

International audienceHereditary pancreatitis (HP) is an autosomal dominant disorder with incomplete penetrance characterized by recurring episodes of severe abdominal pain often presenting in childhood. Although this disorder has only been recently described, about 100 families have been documented worldwide. The pathophysiology of this disorder is unknown. Here, a large French family of 147 individuals (47 of whom were affected) from a four-generation kindred with HP has been examined and a genome segregation analysis of highly informative microsatellite markers has been performed. Linkage has been found between HP and six chromosome 7q markers. Maximal two point lod scores between HP and D7S 640, D7S 495, D7S 684, D7S 661, D7S 676 and D7S 688 were 4.00 (θ = 0.143), 5.85 (θ = 0.143), 4.91 (θ = 0.156), 8.58 (θ = 0.077), 8.28 (θ = 0.060), 4.40 (θ = 0.169), respectively. Multipoint linkage data combined with recombinant haplotype analysis indicated that the most likely order is : D7S 640-D7S 495-D7S 684-D7S 661-D7S 676-D7S 688, with the HP gene situated in the underlined region. As in all families reported in the literature, the clinical presentation of the disease is identical to the presentation of sporadic cases, one could expect that the knowledge of the HP gene could be a clue to pancreatitis in general. Based on its map position, this is the first step towards the positional cloning of the Hereditary Pancreatitis Gene (HPG)

A missense mutation in the alpha-actinin 1 gene (ACTN1) is the cause of autosomal dominant macrothrombocytopenia in a large French family.

Inherited thrombocytopenia is a heterogeneous group of disorders characterized by a reduced number of blood platelets. Despite the identification of nearly 20 causative genes in the past decade, approximately half of all subjects with inherited thrombocytopenia still remain unexplained in terms of the underlying pathogenic mechanisms. Here we report a six-generation French pedigree with an autosomal dominant mode of inheritance and the identification of its genetic basis. Of the 55 subjects available for analysis, 26 were diagnosed with isolated macrothrombocytopenia. Genome-wide linkage analysis mapped a 10.9 Mb locus to chromosome 14 (14q22) with a LOD score of 7.6. Candidate gene analysis complemented by targeted next-generation sequencing identified a missense mutation (c.137GA; p.Arg46Gln) in the alpha-actinin 1 gene (ACTN1) that segregated with macrothrombocytopenia in this large pedigree. The missense mutation occurred within actin-binding domain of alpha-actinin 1, a functionally critical domain that crosslinks actin filaments into bundles. The evaluation of cultured mutation-harboring megakaryocytes by electron microscopy and the immunofluorescence examination of transfected COS-7 cells suggested that the mutation causes disorganization of the cellular cytoplasm. Our study concurred with a recently published whole-exome sequence analysis of six small Japanese families with congenital macrothrombocytopenia, adding ACTN1 to the growing list of thrombocytopenia genes

Morphology of platelet and MK cells from the proband and a control.

<p>(<b>A</b>) Morphology of platelets in the bone marrow smears, which were stained with May-Grunwald Giemsa. The platelet in the proband is much larger than its normal size by reference to the red blood cells and to the control. The analysis was performed on an optical microscope (Leica DMR) with a 50×/1.40 numerical aperture oil objective lens (Leica). (<b>B</b>) Morphology and size (indicated by red arrows in the proband) of platelets from the peripheral blood observed by electronic microscopy (magnification of 10000×). (<b>C</b>) Morphology of a representative cultured MK from the bone marrow observed by electronic microscopy (magnification of 6000×). Ultrathin sections were examined by a JEM-1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV.</p

Evaluation of the functional effect of <i>ACTN1</i> p.Arg46Gln on transfected COS-7 cells by immunofluorescence.

<p>COS-7 cells transfected with either wild-type (top panel) or mutant (lower panel) <i>ACTN1</i> plasmid construction (as well as non-transfected cells) were stained with three different flurochromes as indicated in the Figure. In the merged image, overexpressed α1-actinin, normal F-actin network and counterstained nuclei appear in green, red and blue, respectively. In the top right panel, the arrow indicates an untransfected cell. Fluorescence was visualized on an Olympus BX-61 fluorescence microscope with a 60×/1.40 numerical aperture oil objective lens (Plan Apo, Olympus). Image capture was performed with a Diagnostics Instruments CCD camera system using SPOT Advanced software version 3.2.4 (Diagnostic Instruments, Sterling Heights, MI). Image processing was made with Image G (1.45S, NIH, USA).</p