Band 36, Heft 8

Until recently, the “human genome” programs were mainly directed towards the development of maps to identify disease genes. The genetic map comprises about 8000 highly informative second generation markers of the microsatellite type. The density of markers is now sufficient to localize a gene for a monogenic disease with a precision of 1 to 2 million base pairs easily, and to define intervals which contain susceptibility genes for multifactorial disorders. A third generation map based on single nucleotide polymorphisms that can be genotyped using DNA chip technology is in progress. The physical map, based on sets of overlapping yeast artificial chromosomes ordered using sequence-tagged sites, covers over 90 % of the genome. However, this physical map cannot serve as a support for sequencing because of the numerous rearrangements that occur in yeast artificial chromosomes. An international network of laboratories has mapped a set of more than 30000 expressed sequences from cDNAs using whole genome radiation hybrids that enable integration of genes within existing maps. The human genome program is now progressively shifting to massive sequencing, although sequence ready maps are not available for the major part of the human genome. Similarly, our capacity to interpret the available genomic sequence remains limited.

The Human Genome Project, the mapping of our 100000 genes and the sequencing of all of our DNA, will have major impact on biomedical resarch and the therapeutic and preventive health care. The tracing of genetic diseases to their molecular causes is rapidly expanding diagostic and preventive options, while the increased insights into molecular pathways open tremendous perspectives for pharmacological and genetic therapies. The design of animal model systems for the functional study of disease and development of bioinformatics and biostatistics to improve our pattern recognition abilities are greatly accelerating progress. However, the optimal value from the current explosion of ‘data mining’ possibilities will only be gained when the basic data are made and kept publicly accessible, at the same time preventing the jeopardisation of the protection of intellectual property, arising from downstream inventions. This is one of the goals of HUGO, the international Human Genome Organisation, established 9 years ago to assist coordinating data acquisition and exchange and societal implementation of the genome project. Additional points of major importance in this historic endeavour are the safe-guarding of a worldwide balance in the contribution and benefits to countries and population, the prevention of stigmatisation and discrimination of individuals and groups and the maintenance of respect for the priceless diversity of our world's cultures and traditons.

Detection of mutations in genes is vital throughout biology, however, this activity is time-consuming, expensive and requires a high degree of skill. This is unsatisfactory in a field which is increasing importance. Around 10–12 methods are commonly used with some predominating. All have their advantages and disadvantages and none is perfect. Sequencing is said to be the gold standard for detecting new mutations (and must be used to define it), but six or so methods have been described to make the search quicker to avoid sequencing the whole gene. These are called scanning methods. Other methods are used to detect known mutations and referred to as diagnostic methods. These methods will be briefly reviewed. Once mutations are described, they are usually published. The mutation lists are collected for convenience, analysis or research. In recent years, it has been realised that these lists are vital for research, patient care and commercial activities. These activities will be reviewed and the co-ordinating role of the HUGO Mutation Database Initiative will be outlined.

The family has proven the most appropriate unit with which to study Mendelian diseases. There are, however, certain limitations on the use of the family as a fundamental unit in the study of common diseases, most of which are complex genetic diseases. The groups that are most likely to yield the genetics of complex diseases are isolated populations with strong founder effects. Therefore, access to such populations is proving to be a precious resource in the work on the genetics of common diseases. The Icelandic population is an excellent population for the study of the genetics of common diseases; it is genetically homogeneous, with founder effects for many traits, and the genealogy of the entire nation is well documented back to the founding days. Furthermore, the nature of the Icelandic national health care system facilitates the assignment of phenotypes in the search for disease genes. Decode Genetics has begun to study of the genetics of 20 of the most common diseases in the Western parts of the world. The company has placed the groundwork for the construction of an encrypted database with information on the health care of the entire nation, genealogy of the entire nation, genotyping information with high density of markers on a large part of the nation (including typing for known disease genes), and resource use in the Icelandic health care system. The plan is to build the database with approval of participating individuals as well as Icelandic government and health care officials. The database will be used to model health care as viewed in the context of genetic predisposition to the development of disease. The database will also be used in the search for drug targets in complex diseases and in the solution of pharmacogenomic problems. Basing the company in Iceland directly benefits the population in terms of employment and return on investment as well as providing the health care system with an information resource which may be used in preventive medicine and in the optimization of health care in Iceland.

Gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease such as restenosis after angioplasty, vascular bypass graft occlusion, transplant coronary vasculopathy, homozygous familial hypercholesterolemia and cystic fibrosis, for which no known effective therapy exists. Gene therapy requires efficient in vivo gene transfer technology. During the past decade, many gene transfer methods including viral transfer techniques have been developed, and some are being applied clinically in human gene therapy studies. Molecular biology and pathophysiology of the cardiovascular system have started to emerge, and the time is ripe for the introduction of gene therapy to the management of cardiovascular disorders. In this review, we have focused on the future potential of oligonucleotide-based gene therapy for the treatment of cardiovascular disease.

Research over the last decade has provided us with much information about the mechanisms that integrate signal transduction pathways with specific gene expression programs. We discuss the types of mechanisms used by different pathways to modulate transcription factor activity, citing examples from diverse molecular systems. Careful regulation of these pathways is essential to maintain balanced transcriptional control. Understanding the mechanisms that control gene activity will enable us to intervene therapeutically in diseases associated with deregulated transcriptional activity.

Antisense oligonucleotides were used to demonstrate the importance of insulin-like growth factor II and α-fetoprotein for the growth of hepatoma cell lines. The level of insulin-like growth factor II was found to correlate positively with cell proliferative activity, whereas α-fetoprotein was not. We have developed an in vitro system for the screening of antisense oligonucleotides effective for inhibiting target protein production. Using this system, the effectiveness of antisense oligonucleotides can be determined even when a specific antibody or activity assay method is not available. These approaches will be useful for verifying the physiological role of other oncoproteins or proteins in living cells, and antisense oligonucleotides may be developed as new therapeutic agents.

At the risk of ignoring the benefits, the problems of genetic testing have been widely discussed. Many clinical laboratories are archival stores for human tissues and very few have formal policies and procedures for the use of this material. There is little evidence of planning or preparation of appropriate protocols for the safekeeping of the collected clinical material. The process of informed consent needs to be re-examined. The standard of what would be expected by a reasonable and prudent person poses challenging questions relating to confidentiality and privacy, control and ownership of the stored tissue and cells, withdrawing from a research study, length of storage of samples, the use of biological materials by other parties, and the use of biological materials for purposes other than those for which they were first obtained. There also needs to be a clear understanding of identifiable, non-identifiable and anonymous samples. Clinical laboratory staff and their professional organisations need to engage in the discussion and development around the ethical, legal and social issues raised by testing human DNA. Clinical research laboratories will be increasingly involved in DNA testing and will be asked for access to stored blood or tissue. It is essential that professional responsibility is understood and is then exercised in the presence of appropriate policies and procedures. There is also a paramount need to involve the public who are already demonstrating evidence of alienation from the world occupied by genetics research.

During the last few years, an important development of molecular biology techniques has been observed in research and clinical laboratories, and consequently the availability of DNA is becoming essential for epidemiological studies. Several DNA extraction procedures have been proposed. However as the quantity and quality of the DNA extracted is very variable, standardization of the storage of samples, and of extraction procedures becomes essential. Three steps will be considered. (i) Procedures of whole blood preservation prior to extraction which seem to affect yield and purity of the DNA extracted; (ii) DNA extraction procedures, which must be validated by a systematic DNA quality control using DNA molecular weight screening and spectrophotometric analysis, and also by verification that the DNA obtained is well adapted for molecular biology applications; (iii) the third important factor to study is the storage of DNA, since data on short-term (several months) and especially on long-term (several years) DNA stability is often missing and/or conflicting. Detailed studies must be done in order to establish guidelines for proper handling of genetic material.

A key issue in nucleic acid-based assays related to infectious disease testing and genetic testing is the sample preparation. This has to be convenient, efficient and suitable for automation. We describe a new method based on the use of glass magnetic particles. The steps involved are the lysis of bacterial cells and viral particles by chaotropic salts, binding of released nucleic acids to the magnetic glass particles, the removal of inhibitors and the release of purified nucleic acids ready for PCR amplification. The method fulfills most of the requirements for sample preparation such as sensitivity, universality and suitability for automation.

In our efforts to develop diagnostic tests for complex multifactorial disorders, and to assist the research community in evaluating genetic markers for predisposition to cardiovascular disease, we have developed a prototype assay to genotype up to 35 variable sites among 15 genes. The candidate markers in this panel were selected from biological pathways likely to contribute to the development and progression of cardiovascular disease. Each sample is amplified in two multiplex polymerase chain reactions that are then hybridized to an array of immobilized oligonucleotide probes. The assay has been applied to a population-based cohort representing 238 families; allele frequencies observed among 455 unrelated parents from this cohort agree with available literature values. Data from a cohort of 142 lipid-clinic patients were used to explore locus associations with arterial occlusion, as measured by quantitative angiography. This prototype assay provides a research tool for studies to assess the association of multiple markers with disease, and for clinical studies to evaluate marker association with patient responsiveness to experimental therapies.

Germline mutations in the adenomatous polyposis coli gene cause familial adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5′-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.

Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, coamplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.

The combination of reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis has been proposed for the quantitative determination of ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase subunits M and H, and cyclooxygenase 1 and 2. We designed fluorescence labeled common PCR primers in the sequences highly homologous between two subunit and isozyme cDNAs, and performed reverse transcribed-PCR and fluorescencebased single strand conformation polymorphism analysis. PCR efficiency was almost the same for the different target sequences, so analysis of mixtures of known amounts of lactate dehydrogenase M and H revealed linear and precise proportions of lactate dehydrogenase M mRNA. It was shown that template concentrations and number of PCR cycles did not affect the determination of proportions of lactate dehydrogenase M to total lactate dehydrogenase. The procedure was applicable to a determination of cyclooxygenase-2 proportion; furthermore, the present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.

Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used.

In this article, we describe a useful modification of the polymerase chain reaction for amplification applicable to hepatitis C virus genotyping and determination of its subtypes. The method is fast, cheap and simple for detection of any known point mutation, and could be used in every laboratory with experience in polymerase chain reaction technique. We could differentiate hepatitis C virus subtype 1b from other subtypes and 2b from 2a and other subtypes as well. We could also differentiate hepatitis C type 3 using a type-specific oligonucleotide from 3a subtype, thus covering the most common hepatitis C virus (sub)types present in the European region.

In this paper, we introduce two autoradiography imaging systems from Packard Instrument Company, that provide unmatched speed, accuracy, sensitivity, and resolving power for radioisotopic imaging in biomedical research. The first system is a direct nuclear imaging system, which provides fast and accurate nuclear counting without intermediate steps. Data are displayed in real time so that counting, imaging and data analysis can be done all at the same time. The second system is a new storage phosphor system that was designed with a unique optical assembly which provides high resolution imaging. With various types of phosphor screens, one can optimize the performance of this storage phosphor system with any radiolabel and any appropriate sample size. With these two systems, quantitative radioisotopic imaging becomes not only faster and easier, but also more consistent and accurate, providing researchers with a higher standard in molecular biology imaging.

The Kirsten- ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras -mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c- erbB -2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, “enrichment PCR” must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5′ primer (…GGA C̱CT GGT…) for an enzyme ( Bst NI) (5′CC!WGG 3′) to differentiate between wild-type sequence (…GGA G̱CT GGT…) (during amplification, the G̱ is replaced by a C̱) and mutated sequences (̱…GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the secound round altogether. The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.

The performance of an automated DNA amplification assay (Roche Cobas ® Amplicor™ Mycobacterium Tuberculosis Test (aPCR) was compared with an in-house single tube nested polymerase chain reaction (nPCR) for the direct detection of Mycobacterium tuberculosis in respiratory specimens. Among 385 specimens, 56 were culture positive for mycobacteria (44 positive for Mycobacterium tuberculosis and 12 positive for nontuberculosis mycobacteria). The diagnostic sensitivities of aPCR and nPCR were 86 % and 91 % whereas a 100 % diagnostic specificity of both assays was attained. By aPCR, inhibitors were detected in 6% of the clinical samples. For nPCR, the usage of a new thermostable DNA polymerase facilitated pre-PCR decontamination using uracil-N-glycosylase and “hot start” in single step procedure. The results of the study indicated that DNA amplification assays, either manual or automated, were rapid and specific tools for diagnosing pulmonary tuberculosis.

The Amplicor™ HBV Monitor Test for quantitative detection of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates. Evaluation in a routine diagnostic laboratory showed excellent sensitivity and adequate reproducibility; however, a more automated format would be desirable. The Amplicor™ HBV Monitor Test is useful for recognizing those patients who might benefit from antiviral treatment and for evaluation of the efficacy of anti-hepatitis B virus treatment.

The etiology of cardiovascular diseases is known to be multi-factorial. Some forms of cardiovascular disease are influenced by unclear genetic factors but are predominantly affected by factors such as diet, obesity, cigarette smoking, diabetes mellitus and dyslipidaemia. Some are caused by specific gene defects, with environmental factors playing a precipitating role. Others result from complex gene-gene or gene-enviroment interactions. Advances in knowledge of the molecular genetics of lipidaemic and vascular disorders have identified gene aberrations that are associated with cardiovascular disease. Techniques in molecular biology have been applied for rapid and reliable detection of specific gene defects to provide unequivocal diagnosis beneficial for appropriate drug therapy and genetic counseling. Pre-symptomatic diagnosis is possible and carriers can be advised on effective preventive measures. However, prior to the provision of a molecular diagnostic service, all gene alterations associated with cardiovascular disease have to be identified and their prevalence established in a population. The number of mutations in so many causative genes is enormous. While more cost-effective laboratory methodologies will be developed in the future, it is also anticipated that more mutations with direct or indirect effects on cardiovascular disease will be discovered in different populations.

The influence of apolipoprotein E polymorphism and apoE level on serum lipids and apolipoproteins was investigated in 71 healthy people and 43 patients with coronary artery disease from Shanghai. The frequency of apoE alleles was 0.06 for ε2, 0.86 for ε3, and 0.07 for ε4 in the healthy group, and 0.14 for ε2, 0.77 for ε3, and 0.09 for ε4 in the coronary artery disease group. There was no significant difference in the frequency of apoE alleles between these two groups. Serum levels of triglyceride and apo AI did not differ according to apoE genotypes, whereas serum level of apoB was significantly different according to apoE genotypes (p<0.05) both in healthy and coronary artery disease groups. However, in the healthy group, apo ε2 allele carriers had significantly higher level of apoE than apo ε3 and ε4 allele carriers (p<0.001) and apo ε4 allele carriers had significantly higher level of total cholesterol than apo ε3 and ε2 allele carriers. These were not observed in the coronary artery disease group. ApoE concentration was positively correlated with cholesterol, apoAI, and apoB levels in the control subjects and no significant correlation was observed with triglyceride level. In contrast, apoE level was positively related only to triglyceride level in the coronary artery disease group. In the control group, apoE genotypes and apoE level explained together 19.3 % and 26.6 % of the variability of apoB and cholesterol level, respectively, apoE polymorphism explained 23 % of the variability of apoE level and apoE level explained 13.2 % of the variability of apoAI level. In the coronary artery disease group, only apoE level explained 41.7 % of triglyceride variability. Finally we compared our results with those previously obtained in a French healthy population, the Stanislas cohort. Results suggested that there were some difference between the Chinese control and the French subjects.

Angiotensin converting enzyme is a key component of the renin angiotensin system that plays an important role in cardiovascular regulation. It seems to modulate cardiovascular growth by virtue of its role in the conversion of angiotensin I to angiotensin II and degradation of kinins. A deletion polymorphism localized in intron 16 of the human angiotensin converting enzyme gene, corresponding to a 287 bp long Alu repetitive sequence, was found to be associated with increased risk of myocardial infarction in various subgroups, including European, French and Japanese coronary patients. This angiotensin converting enzyme gene I/D polymorphism was examined by the polymerase chain reaction in a cross-sectional study of 201 healthy Indian subjects and 150 patients (angiographically proven cases of coronary artery disease) whose serum angiotensin converting enzyme levels were concomitantly measured. The D/D, I/D and I/I genotypes were found in 20.66 %, 46.66 % and 32.66 % of the Indian coronary heart disease patients and in 23.38 %, 49.75% and 26.86 % of controls respectively. One of the reasons for not finding an association between the D allele and coronary artery disease in this study could be the ethnic heterogeneity and disease status heterogeneity among the patients and controls. However the phenotypic variance of serum angiotensin converting enzyme levels is strongly influenced by this polymorphism. In the Indian population, the angiotensin converting enzyme gene I/D polymorphism is not associated with risk for coronary artery disease although it is associated with plasma angiotensin converting enzyme activity. Hence the angiotensin converting enzyme gene I/D polymorphism does not seem to be a useful marker for coronary artery disease in the Indian population.

The missense mutation in the 677th nucleotide (C677T) of methylenetetrahydrofolate reductase gene causes substitution of valine (V) for alanine (A) resulting in three genotypes VV, VA and AA. The VV genotype causes hyperhomocysteinemia and may be a risk factor for coronary artery disease. We determined genotypes by polymerase chain reaction and subsequent restriction fragment length analysis and compared them in 84 patients with type 2 diabetes and in 115 non-diabetic subjects with and without coronary disease. Fractional urinary excretion rate of albumin was assessed by nephelometry. The VV, VA, and AA frequencies in the diabetic and in the control groups were 0.095, 0.357, 0.548 and 0.061, 0.417, 0.522, respectively (p = NS, diabetic vs. controls, χ 2 test). Genotype frequencies did not differ in either diabetic or control subjects between those with or those without coronary disease (χ 2 test). The fractional urinary excretion rate of albumin (mean ±SD) in diabetic patients with the VV genotype i.e. 1.59 ± 0.71 was lower (Kruskall-Wallis test p = 0.002) than in the other genotypes i.e. VA 5.98 ± 9.75 and AA 3.75 ± 4.77, respectively ( post-hoc Mann-Whitney test VV vs. VA p = 0.005 and VV vs. AA p = 0.054, respectively). We found that in patients with type 2 diabetes the methylenetetrahydrofolate reductase VV genotype was associated with a low urinary albumin excretion but not with coronary artery disease or diabetes per se .

We have examined a group of North American subjects, selected to include individuals with a wide variety of HDL-cholesterol concentrations for: 1) mutations in the genes coding for cholesteryl ester transfer protein and hepatic lipase, 2) apolipoprotein E genotype, 3) total cholesterol and triglycerides, 4) HDL-triglycerides. Cholesteryl ester transfer protein activity was also estimated, using a novel technique that does not require separation of substrate and product. Transfer activity was shown to have a monophasic distribution, with a mean activity of 21 pmol substrate transferred/3 h/μl plasma. The cholesterol ester transfer activity of the group with HDL-cholesterol >1.60 mmol/l was significantly less than those with HDL-cholesterol <1.60 mmol/l. The cholesteryl ester transfer protein G1533A mutation was detected at an overall allele frequency of 2.91 %. The mutation was more frequent in the group with HDL-cholesterol <1.60 mmol/l than in those >1.60 mmol/l. It was also more frequent in those with protein activity > 30 pmol/3h/μl plasma than in those with activity <30. These data suggest that this mutation in cholesteryl ester transfer protein is associated with increased transfer activity and reduced HDL-cholesterol concentrations. The cholesteryl ester transfer protein activity assay described here is simple and convenient. Subject to further evaluation and correlation with the present labour and time intensive assays, this commercially available assay offers the potential of rapid, simple analysis of large numbers of samples.

Alcohol dependence, and the medical conditions which arise from prolonged excessive alcohol use, have no single cause. Like other complex diseases, they result from a combination of social, personal and genetic contributions; but within any society genetic variation has a substantial influence on individual risk. The genes presently known to affect alcohol dependence produce variation in alcohol metabolism; other genes which affect personality or susceptibility to intoxication are likely to be significant but so far reproducible evidence is scanty. Designs which include related subjects have advantages for the study of complex diseases, because any association effects can be placed in the context of overall heritability and because linkage analysis can also be included. Examples of our studies of alcohol metabolism, consumption and dependence are presented.

Distinction between benign and malignant T-cell lymphoproliferative diseases can be difficult using morphological criteria. Using multiplex polymerase chain reaction system we have tested a series of patients with various lymphoproliferative disorders to detect clonal T-lymphocyte populations. Results show that clonal amplification products were obtained from all 10 patients with T-cell lymphoproliferative disorders while the amplification of DNA samples from B-cell neoplasms and normal individuals revealed polyclonal amplification products. By splitting the multiplex primer mix, the patient specific T-cell receptor γ rearrangement was determined: five out of ten patients showed the exclusive presence of a single T-cell receptor γ gene rearrangement. Three patients exhibited two rearranged T-cell receptor γ genes, while in two patients positive reactions were obtained with three pairs of primers for variable and joining segments. Molecular analysis of rearranged T-cell receptor genes by multiplex polymerase chain reaction represents a useful and rapid tool for confirming diagnosis, to determine the extent of disease and to monitor the response to therapy.

We have shown the impact of molecular diagnostics related to mutation detection in an extensive family with a strong history of colorectal cancer. The nature and presentation of the cancers suggested that hereditary nonpolyposis colorectal cancer was the most likely cause. The strategies employed have enabled the detection and characterisation of the causative mutation in the proband and predictive testing in the remaining relatives where requested. Using the chemical cleavage of mismatches technique and direct sequencing, the MSH2 and MLH1 genes of the proband were investigated. A single base substitution, C→T at nucleotide 350, codon 117, of the MLH1 gene was identified. Across the family pedigree at specific points, 22 other relatives have been tested for the mutation by direct DNA sequencing from genomic DNA. Of the total of 23 patients tested to date, 11 have the mutation. In conjunction with appropriate genetic counselling, this service has clarified the genetic status of many individuals within this family. Predictive information provided prior to the development of symptoms enables individuals to make informed choices regarding disease management and the future, removing the anxiety associated with the unknown.

Approximately ten percent of patients with malignant melanoma have family histories of the disease, suggesting a genetic predisposition. Germline mutations in tumour suppressor p16 gene have been implicated as disease causing mutations in some of the melanoma families. The frequency of families with p16 germline mutations among melanoma prone families varies from eight to fifty percent. The range of the variability is influenced apparently by the number of melanoma affected individuals within the family, as well as by other, yet unidentified factors. Ethnic background is known to determine both the frequency and the nature of germline alterations. Recently, specific mutations in tumour suppressor genes involved in breast cancer and in colon cancer were found at elevated frequency among Ashkenazi Jews. This report describes results of a screening for p16 germline alterations in a collection of Israeli melanoma families. We have analyzed genomic DNA from thirty one Ashkenazi and non-Ashkenazi Jewish melanoma families, as well as from thirty melanoma patients without an apparent family history of the disease. The entire coding region of the p16 gene was screened by single strand conformation polymorphism analysis and direct DNA sequencing. We have detected a number of carriers with the Ala148 Thr polymorphism at the end of the second exon and several instances of 500(G⇒C) substitution at the 3′ untranslated portion of the gene.

We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1 , and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.

The mutant of CYP2D6*3 allele with A 2637 deletion in exon 5 and the mutant of CYP2D6*4 allele G 1934 →A, splice site defect are among the most common polymorphic alleles of CYP2D6 gene, resulting in a decreased or no activity of CYP isoenzyme. In this study, a reliable polymerase chain reaction-restriction fragment length polymorphism method for identification of CYP2D6*3 and CYP2D6*4 alleles was used to investigate the genotype and phenotype prevalence in the groups of normal controls, and of cirrhosis and cancer patients. The results showed none of 36 controls genotyped for 2D6*3 and 2D6*4 allele to have the 2D6*3 allele with frameshift mutation in exon 5, while 33 % (n=12) were found to bear the 2D6*4 allele with G to A mutation at the intron 3—exon 4 junction. In breast cancer patients (n=35) genotyped for 2D6*3 and 2D6*4 alleles, none with 2D6*3 allele was found either, but 60 % (n=18) were found to bear the 2D6*4 allele. In patients with head and neck squamous cell cancer, there was only one subject with 2D6*3 allele and he was heterozygous. Among them, as many as ten (40 %) patients were found to bear 2D6*4 allele. In the cirrhosis group, none of the patients was found to have the 2D6*3 allele, while the CYP2D6*4 allele was found in 23 % (n=6) patients. The phenotype predicted according to the genotype was as follows: in the control group, 3% of individuals were identified as poor metabolizers, 70 % as extensive metabolizers, and 27 % as heterozygote extensive metabolizers. In the group of breast cancer, 7% of the patients were identified as poor metabolizer, 57 % as extensive metabolizer and 36% as phenotype. In squamous cell cancer and cirrhosis patients, the incidence of poor metabolizer was zero, and of heterozygotes extensive metabolizer 42 % and 31 %, respectively.

We have elucidated the molecular pathology of three types of congenital hypothyroidism. Thyrotropin (TSH) is the major regulator of thyroid function. In cases of isolated congenital TSH deficiency, we found that they are caused by a missense mutation in the conserved CAGYC region of the TSHβ gene. Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), TSHβ genes, and the PIT1 gene itself. In cases of combined deficiency of GH, PRL, and TSH, we found that they are caused by abnormalities in the PIT1 gene, either recessively or dominantly. Sodium dependent iodide symporter (NIS) actively transports iodide into the thyroid cells to produce thyroid hormones. In cases of iodide transport defect, we elucidated that a missense mutation in the transmembrane region of the NIS gene caused them.

We studied a man who sought medical attention at age 28 years because of infertility in both his first and second marriages. His sexual development appeared to have been normal, with normal puberty and virilization, and normal libido and sexual potency. At examination, his testicles were small and soft; otherwise he had a normal physical appearance. Evaluations revealed azoospermia, undetectable in serum before and after 100 μg of intravenously administered gonadotrophin releasing hormone, but moderately elevated lutropin concentration with a brisk rise after gonadotrophin releasing hormone. The α subunit concentration was normal before and after gonadotrophin releasing hormone; that of inhibin B was very low. Analysis of the follitropin β gene, exon 3, revealed a Cys 82 → Arg mutation (TGT → CGT). Judging from studies of the biosynthesis of the chorionic gonadotrophin β subunit one may conclude that inability to form the first intramolecular disulphide bond in the follitropin β subunit results in an abnormal tertiary structure during follitropin β biosynthesis with extensive intracellular degradation of the products, inability to associate with the α subunit and defective glycosylation, as well as inability to form a biologically active hormone. This first male case of follitropin deficiency thus defines a new syndrome of male infertility.

Childhood onset proximal spinal muscular atrophy presents with considerable clinical variability. This study included 14 Croatian children aged 11 days to 8 years with spinal muscular atrophy types I-III verified clinically and electromyoneurographically. DNA of affected children was screened for deletions of exons 7 and 8 of the survival motor neuron gene and for deletion of exon 5 of the neuronal apoptosis inhibitor protein gene. Motor nerve conduction velocity and compound muscle action potential amplitude were decreased in children with spinal muscular atrophy type I and II. Deletions of exons 7 and 8 of the survival motor neuron gene and of exon 5 of the neuronal apoptosis inhibitor protein gene in children with spinal muscular atrophy type I-II suggested existence of more genetic abnormalities as compared to type III. A decrease in compound muscle action potential amplitude and motor nerve conduction velocity in children with spinal muscular atrophy correlated with the disease severity, probably as a result of axonal degeneration. Phenotypic severity in children onset spinal muscular atrophy is directly correlated with the extent of survival motor neuron and neuronal apoptosis inhibitor protein exon deletions.