Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase)

Essential fructosuria is one of the oldest known inborn errorsof metabolism. It is a benign condition which is believed toresult from deficiency of hepatic fructokinase (ketohexokinase,KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolismof dietary fructose, conversion of fructose to fructose-1-phosphate.Despite the early recognition of this disorder, the primarystructure of human KHK and the molecular basis of essentialfructosuria have not been previously defined. In this report,the isolation and sequencing of full-length cDNA clones encodinghuman ketohexokinase are described. Alternative mRNA speciesand alternative KHK isozymes are produced by alternative polyadenylationand splicing of the KHK gene. The KHK proteins show a high levelof sequence conservation relative to rat KHK. Direct evidencethat mutation of the KHK structural gene is the cause of essentialfructosuria was also obtained. In a well-characterized family,in which three of eight siblings have fructosurla, all affectedindividuals are compound heterozygotes for two mutations Gly40Argand Ala43Thr. Both mutations result from G     

Detection of disseminated tumor cells in the lymph nodes of colorectal cancer patients using a real-time polymerase chain reaction assay

Introduction Postoperative treatment for colorectal cancer depends on tumor stage as defined by the International Union Against Cancer (UICC). Adjuvant chemotherapy is not recommended in patients without lymph node involvement (UICC stages I and II). As many as 20–30% of these patients, however, will develop recurrence. Aims and objectives We conducted this study to determine the presence of disseminated tumor cells in the lymph nodes by quantitative real-time polymerase chain reaction (QRT-PCR) for cytokeratin 20 (CK20) in an attempt to provide supplementary information compared to histopathological findings. Materials and methods Using a standard QRT-PCR assay, we examined primary tumors and 391 lymph nodes from 31 patients with completely resected colorectal cancer. Results Of the 31 primary tumors, 29 were positive for CK20 by QRT-PCR. Discussion An examination of the lymph nodes from the 29 patients with CK20-positive primary tumors revealed that 35 (92.1% sensitivity) of the 38 histopathologically positive lymph nodes and 54 (16.7%) of the 324 histopathologically negative lymph nodes were positive by molecular analysis. CK20 expression was detected in 10 (100%) of 10 patients with a histopathologically positive lymph node status (pN1). In 9 (47.4%) of 19 patients with negative histopathological results (pN0), we detected a CK20 mRNA signal in at least one lymph node. Whereas eight patients with histopathologically negative lymph nodes could be upstaged on the basis of the molecular findings, no patient would be downstaged. Conclusion Our results suggest that QRT-PCR for CK20 is a useful tool for the quantitative detection of micrometastases in the regional lymph nodes. We introduce a standardized procedure that integrates a molecular diagnostic technique in the clinical staging.     

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity

1. The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2. Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-((+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nM, and was 4, 5 and 7 fold more potent than the antagonists [+/-)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3. CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4. In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CA1 pyramidal cell firing rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).     

Ehlers-Danlos syndrome type IV caused by Gly400Glu, Gly595Cys and Glyl003Asp substitutions in collagen III: clinical features, biochemical screening, and molecular confirmation

Three patients with Ehlers-Danlos syndrome type IV (EDS IV) and biochemical evidence of structural defects in collagen III were investigated for mutations within the collagen III gene ( COL3A1 ). Single strand conformation polymorphism analysis of α1(III) cDNA indicated the presence of different heterozygous sequence changes in each of the patients. Nucleotide sequencing revealed mutations leading to the substitution of glycine 400 with glutamic acid, glycine 595 with cysteine, and glycine 1003 with aspartic acid. EDS IV is a life-threatening disorder which, as the clinical histories of our patients and their families show, still often escapes diagnosis. Biochemical and molecular studies can clarify the diagnosis and help provide appropriate management and counselling.     

The kyphoscoliotic type of Ehlers-Danlos syndrome (type VI): differential effects on the hydroxylation of lysine in collagens I and II revealed by analysis of cross-linked telopeptides from urine

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS type VIA) (OMIM 225400) is an autosomal recessive connective tissue disorder that results from mutations in the lysyl hydroxylase 1 gene (PLOD1) causing underhydroxylation of lysine residues in tissue collagens, particularly of skin. Previous studies have shown that the pool of collagen cross-linking amino acids, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) excreted in urine has an abnormally low HP/LP ratio, which is diagnostic of the condition. Here we isolated cross-linked peptides containing these residues from the urine of a child with EDS VIA homozygous for a mutation that results in a stop codon and effective null expression of PLOD1 enzyme activity. Peptides that had originated from bone type I collagen and cartilage type II collagen were identified. A cross-linked N-telopeptide fraction that is derived from bone type I collagen contained only LP, no HP, which means that the helical lysines at residues 930 of alpha 1(I) and 933 of alpha 2(I) of the collagen triple-helix had not been hydroxylated. The equivalent peptide fraction from a normal child's urine gave a ratio of HP to LP of 1.5:1 typical for normal bone collagen. A second cross-linked peptide that is derived from the C-telopeptide domain of cartilage type II collagen showed both HP and LP in a 2:1 ratio, compared with 18:1 for the equivalent peptide from a normal child's urine. The results show that in EDS VIA, bone type I collagen is more markedly underhydroxylated than cartilage type II collagen, at least at those helical sites that form cross-links. The residual fraction of HP found in the urine of EDS VI patients therefore appears to be contributed in significant part by the degradation products of cartilage. Since PLOD1 is null, other PLOD genes must be responsible for the helical hydroxylation activity that results in HP. The presented approach of analyzing urinary cross-linked C-telopeptide fragments of type II collagen may allow the detection of chondrodysplasias due to genetic defects in lysyl hydroxylase isoforms active in cartilage.     

Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene

Mutations in the human fibrillin 1 gene (FBN1) cause the Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Knowledge about FBN1 mutations is important for early diagnosis, management, and genetic counseling. However, mutation detection in FBN1 is a challenge because the gene is very large in size ( approximately 200 kb) and the approximately 350 mutations detected so far are scattered over 65 exons. Conventional methods for large-scale detection of mutations are expensive, technically demanding, or time consuming. Recently, a high-capacity low-cost mutation detection method was introduced based on denaturing high-performance liquid chromatography (DHPLC). To assess the sensitivity and specificity of this method, we blindly screened 64 DNA samples of known FBN1 genotype exon-by-exon using exon-specific DHPLC conditions. Analysis of 682 PCR amplicons correctly identified 62 out of 64 known sequence variants. In three MFS patients of unknown FBN1 genotype, we detected two mutations and eight polymorphisms. Overall, 20 mutations and two polymorphisms are described here for the first time. Our results demonstrate 1) that DHPLC is a highly sensitive (89-99%, P = 0.05) method for FBN1 mutation detection; but 2) that chromatograms with moderate and weak pattern abnormalities also show false positive signals (in all 45-59%, P = 0.05); 3) that the difference in the chromatograms of heterozygous and homozygous amplicons is mostly independent of the type of sequence change; and 4) that DHPLC column conditions, additional base changes, and the amounts of injected PCR products influence significantly the shape of chromatograms. A strategy for FBN1 mutation screening is discussed.