Over the past decade geneticists have searched for rapid and efficient means of identification of genetic diseases. Nucleic acid techniques, such as restriction fragment length polymorphisms, polymerase chain reaction, and sequence analysis of amplified DNA are making increasing inroads as tools in diagnostic laboratories. Our laboratory is involved in the molecular diagnostics of several genetic diseases including Duchenne muscular dystrophy, familial adenomatous polyposis coli, tuberous sclerosis, and osteoporosis. Each disease varies in type of molecular lesions and different approaches are applied to achieve the best mutation detection rate. Duchenne/Becker muscular dystrophy (DMD/BMD) is a lethal X-linked recessive disease caused by mutations within the dystrophin gene (DMD gene). The DMD gene is the largest known human gene, spanning about 2, 500kb and consisting of 79 exons. In approximately 60% of DMD/BMD patients, deletions of one or more exons are detected. Carrier detection still causes problems for many cases, because of the enormous size of the DMD gene and the high intragenic recombination frequency. Familial adenomatous polyposis coli (FAP) is a dominantly inherited autosomal disorder caused by germ line mutation in the adenomatous polyposis coli (APC) gene and is characterized by early onset of multiple adenomatous polyps, which leads to the development of colorectal carcinoma. The development of colon carcinoma is associated with loss of heterozygosity (LOH) in the APC gene and accumulation of mutations in a number of tumor suppressor genes. Detection of mutation carriers in FAP families before pathological symptoms occur is very important and makes possible clinical treatment. Molecular changes in the APC gene were found in 30% of the studied patients with familial adenomatous polyposis coli, with a similar frequency for the common deltal 309 mutation as in other populations. In a Polish population of FAP patients, most mutations were localized in a region of the APC gene encompassing colons 1040-1309.
Tuberous sclerosis (TSC) is an autosomal dominant disease with variable penetrance, characterized by alterations in various organs including brain, heart, and kidneys. In about 2/3 of patients a family history of TSC is not observed. TSC is caused by a mutation in one of two genes—TSC1 or TSC2. Discovered recently, the TSC1 gene is composed of 23 exons; the first two exons are not expressed and exon 2 is alternatively spliced. Analysis of mutations was performed for patients, and mutations were identified, and found to be distributed in different exons. Approximately 75% the mutations could be detected by PCR-single-strand conformation polymorphism (SSCP) and PCR-heteroduplex (HD). All mutations identified in the group of patients led to premature termination of translation. Experiments performed over the last few years allowed the identification of three novel genes located in the 9q34 region (TSC1, RalGDS, and XPMC2H) and the characterization of structure, level of expression, and site of expression of the TSC1 gene. Osteoporosis is a common human disease and is under genetic control, with several genes most likely contributing to the development of the disorder. One of many gene candidates that could potentially regulate bone development is the osteoprotegerin (OPG) gene. Using PCR-SSCP and PCR-HD techniques, we investigated sequence variations in the human OPG gene in DNA samples from patients with osteoporosis and osteopenia and healthy controls. From seven detected polymorphisms five were intronic substitutions, IVS1+15C→T, IVS2+4C→T, IVS3-5C→T, IVS4-24C→A, IVS4+8A→C; one represented a two base pair deletion, IVS3+46delTC; and the last polymorphism, 9G→C, detected in exon 1 of the OPG gene, was associated with osteoporosis. The relative risk for osteoporosis is estimated to be 2.99 in individuals who carry allele C (95% confidence interval 1.06-8.41).
We have reviewed recent progress in clinical as well as molecular aspects of familial Parkinson's disease (PD). Three genes have been identified as causes for different forms of familial PD or parkinsonism, i.e., alpha-synuclein, parkin, and tau. In addition, 9 other chromosome loci were identified to be linked to familial PD or dystoniaparkinsonism. Alpha-synuclein mutations cause autosomal dominant levodopa-responsive PD. Parkin mutations cause autosomal recessive young onset familial PD. Tau gene mutations cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Mutated alpha-synucleins showed an increased tendency for self aggregation into an anti-parallel beta-sheet structure. Parkin protein appears to be essential for the survival of pigmented nuclei of the brain stem. Tau protein is an important microtubule-associated protein. Elucidation of the molecular mechanism of nigral neuronal death due to these mutations will contribute to a better understanding of familial as well as sporadic PD.
We screened for germline mutations of the mismatch repair genes and related genes in 37 Japanese hereditary nonpolyposis colorectal cancer (HNPCC) cases. Fifteen kindreds fulfilled the Amsterdam criteria; and 22 other kindreds, the less stringent “Japanese clinical criteria.” When we examined the entire coding sequences and flanking intronic sequences of the hMLH1 and hMSH2 mismatch repair genes by PCR-SSCP, 9 of the 15 (60%) cases satisfying the Amsterdam criteria and 3 of the 22 (13.6%) cases according to the Japanese clinical criteria showed germline mutations, indicating a significant difference in the detection rate. Interestingly, the mutation frequency of hMSH2 (11/12, 91.7%) was much higher than that of hMLH1 (1/12, 8.3%). We also analyzed colorectal cancer-related genes, e.g., the transforming growth factor-β type II receptor (TGF-βRII) gene, in several microsatellite instability (MSI)-negative HNPCC cases. A substitution of methionine for threonine at colon 315 in the kinase domain of TGF-βRII gene was found in one kindred with the Japanese clinical criteria. Thus, it is likely that causative genes of HNPCC are heterogeneous. MSI has been reported in familial gastric cancers (FGC). However, genetic defects responsible for this phenotype, that is, mutations in mismatch repair genes such as hMLH1 and hMSH2 have not been detected in most FGC cases. Earlier studies have shown that the promoter region of the hMLH1 gene was methylated in some sporadic colorectal cancers. To determine how FGC acquire MSI, we examined the MSI status, hMLH1 protein expression, and methylation status of the hMLH1 promoter region in FGC cases. Out of 9 cancers from 8 FGC kindreds, 6 showed MSI at one or more loci; no germline mutations in the hMLH1 or hMSH2 genes were detected; 4 cancers exhibiting MSI displayed aberrant hMLH1 expression. Methylation in the hMLH1 promoter region was found in these 4 cases. In contrast, the cancers displaying hMLH1 protein expression were not methylated in the hMLH1 promoter region. Our data show a significant association between the absence of hMLH1 expression and methylation of its promoter in FGC cases with MSI. This suggests that the mechanism of inactivation of hMLH1 is epigenetic and that there are other genes responsible for FGC.
Dinucleoside 5′, 5′′′-Pα, Pω-polyphosphates have been found in all cells examined. So far, the only known specific enzyme that catalyzes the synthesis of these polyphosphates is GTP:GTP guanylyltransferase. This enzyme produces Gp4G, Gp3G, and various Np3-4Gs. The adenylated counterparts, such as Ap3A and Ap4A, are synthesized by ligases, at least by some aminoacyl-tRNA synthetases. Ap4A phosphorylase, firefly luciferase, acyl-CoA synthetase, and the DNA- and RNA-ligases are also able to produce Np3-6As. By contrast, in addition to nonspecific enzymes such as phosphodiesterase I and nucleotide pyrophosphatase, there occur in all types of organism specific enzymes that degrade Np3-6N's. These are the Np3N′ hydrolases, the asymmetrically and symmetrically acting Np4N′ hydrolases, the NpnN′ phosphorylases, and recently described hydrolases that prefer Ap6A and Ap5A as substrates. Human Fhit protein, a putative tumor suppressor, behaves as a typical Np3N′ hydrolase and is a member of the HIT (histidine triad) protein family. The human Np4N′ hydrolase belongs to the MutT motif or Nudix (nucleoside diphosphate attached to “x”) hydrolase protein family. The level of Np3-6N's in the cells increases dramatically under stress. It is suggested that these minor nucleotides act as both intra- and extracellular signalling molecules. ApnAs interact with purine receptors and affect vascular tone. Chemically synthesized analogues of NpnN's help us to understand the mechanism of action of the enzymes mentioned, and some of them are candidates for drugs.
The human nuclear genome is now being extensively analyzed in search for single nucleotide polymorphisms (SNPs) that are useful for predicting susceptibility to various diseases. To identify SNPs associated with diseases or longevity in the human mitochondrial genome, we have started analyzing the entire sequences of mitochondrial DNA (mtDNA) from 576 individuals, including 96 centenarians, 96 patients with Parkinson's disease, 192 patients with diabetes mellitus with or without severe vascular complications, and 192 young adults with or without obesity. We are going to establish a database for SNPs in the human mitochondrial genome based on these analyses. We assume that the SNPs in the mitochondrial genome are functionally as important as those in the nuclear genome. We hypothesize that the functional property of mitochondria of an individual is genetically influenced by a combination of common, rare, and individual variations in their mtDNA. The construction of human mtDNA database will provide important information for developing an efficient multiplex analysis system for SNPs to predict longevity or susceptibility to age-associated diseases.
Interaction of integrin receptors with ligands is critically involved in a number of physiological processes and in the pathogenesis of many human diseases. However, the mechanism of ligand recognition is still poorly understood. Macromolecular protein ligands such as fibrinogen have several contact sites which, after initial docking into the activated integrin receptor, stabilize the receptor-ligand complex so that it becomes nondissociable. Available data on integrin-ligand interactions on cells indicate that activation does not result in a change in the thermodynamics of this reaction, but does increase the kinetics of the interaction. The ligand binding to integrins has been previously described kinetically to be a two-step reaction, leading through the initially reversible complex to the final irreversible one. The fibrinogen binding to αIIbβ3 induces conformational changes in the receptor molecule that lead to platelet aggregation and granular secretion, tyrosine phosphorylation of intracellular proteins, and ion fluxes across the membrane. The spatial distribution of contact sites on the receptor for both fibrinogen binding motifs, RGD and HHLGGAKQAGDV (γ400-411), has been characterized by fluorescence resonance energy transfer measurements. Binding experiments using functional analogues of these fibrinogen sequences, cRGD and cHarGD, and fluorescence resonance energy transfer analysis showed that both peptides simultaneously interacted with distinct sites on αIIbβ3 separated by 6.1±0.5nm. However, they were not fully specific and to some extent mutually displaced each other. Binding of cRGD and cHarGD to αIIbβ3 resulted in distinct conformational alterations in the receptor and the opposite microenvironmental changes, as indicated by exposure of ligand induced binding site (LIBS) epitopes and electron paramagnetic resonance analysis using 5-doxylstearic acid as a spin probe, thus supporting the concept that both peptide ligands, due to binding to distinct sites, initiate the sending of different signals during outside-in signaling mediated by αIIbβ3. These data suggest that during fibrinogen-αIIbβ3 complex assembly, the sequential contacts between key sequences of each component of the complex may initiate different waves of conformational changes in the receptor transmitted into the cell. Furthermore, present data suggest that binding of different macromolecular ligands to αIIbβ3, such as fibronectin or von Willebrand factor, both containing only the RGD motif, may induce distinct signaling pathways from that initiated by fibrinogen.
Glycosylation represents a major posttranslational modification reaction of proteins and plays a pivotal role in a variety of cellular events. N-Acetylglucosaminyltransferases III (GnT-III) and V (GnT-V), and α 1-6 fucosyltransferase are key enzymes in the processing of N-linked glycoproteins, and are heavily involved in cancer progression and metastasis. Very recently we also found that GnT-III is capable of reducing or masking the expression of the αGal epitope, which is a major xenoantigen in swine to human xenotransplantation. In this minireview we focus on the role of the above glycosyltransferases in cancer metastasis as well as in xenotransplantation.
Human prostatic acid phosphatase (EC: 220.127.116.11) belongs to the group of enzymes that can split simple phosphate esters, nucleotides, and phosphoamino acids in phosphoproteins including phosphotyrosine derivatives. The native phosphatase is a glycosylated homodimer with a molecular weight of 100kDa. Specific inhibition and denaturation-renaturation studies showed that the catalytic activity of the enzyme depends on the formation of the dimer. The enzyme is androgen-regulated and is a prostate-specific protein; and its level is often elevated in the blood serum of prostate cancer patients. Prostatic acid phosphatase can be stabilized against various denaturation agents by cross-linking techniques and can be used in different biomedical and biotechnological processes.