Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.     

Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.     

A High-Resolution Interval Map of the q21 Region of the Human X Chromosome

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.     

HPLC-analysis of algal pigments: comparison of columns,column properties and eluents

The effects of columns (Nucleosil C₁₈ODS, MZ-PAH, YMC-PACK C₃₀), column properties (inner diameters of 4 mm, 3 mm and 2 mm, pore-width 10 nm and 30 nm) and eluents (methanol, acetonitrile, acetone, water) were tested on the separation of algal pigments. The length of columns was 250 mm and particle size was 5 μm. Flow rates and gradients were adjusted to optimize peak separation; remaining chromatographic conditions were kept constant. The resolution of chromatographic systems was tested with pigment standards and various algal cultures. Total flow rate and retention times decreased with decreasing inner diameter, whereas pressure, sensitivity and peak-width increased. Pore width had negligible effects on the chromatographic separation of pigments under the test conditions. Only with acetonitrile as eluent were all the taxonomically important pigments resolved adequately: zeaxanthin (Cyanophyceae), lutein (Chlorophyceae), fucoxanthin (Bacillariopyceae), alloxanthin (Cryptophyceae), peridinin (Dinophyceae).     

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

Characterization of the high affinity heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP) and its enhancement by zinc(II)

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP₆₉₅ was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP₆₉₅ which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein.