Carrier analysis and prenatal diagnosis of haemophilia A in North India

The feasibility of DNA diagnosis for haemophilia A in North India was evaluated using intragenic polymorphic DNA markers in factor VIII gene for linkage analysis as well as direct detection of inversion mutation in intron 22 of the gene. The informativity of RFLP (HindIII, BclI and XbaI) and STR (introns 13 and 22) markers for linkage analysis in factor VIII gene was determined in 100 normal individuals. The observed heterozygosity for RFLP markers HindIII, BclI and XbaI was 0.63, 0.60 and 0.48 while that of STR markers introns 13 and 22 were 0.60 and 0.40 respectively. Six and four alleles were identified for introns 13 and 22 and the most frequent allele was 13(CA)26 and 22(AG)n(GT)26 with an allele frequency of 0.53 and 0.62 respectively. The heterozygosities observed for RFLP markers was higher (>70%) than the STR markers (50%) in the affected families with haemophilia A. Inversion mutation was detected in 37% of severely affected patients. Based on present and previous studies from India, a strategy has been proposed to provide molecular diagnosis to a large number of undiagnosed cases of haemophilia A.     

血友病B的基因诊断

The haemophilia B(HB)is caused by the mutations in the factⅨ gene, and is known as an X-linked recessive disease. At present, due to lacking of the eradicative therapy, the gene diagnosis and detection of the carriers is an effective method to prevent the infant patients to be born, block the transmission of harmful gene, and improve the population quality. This should be actively encouraged for its pratical usefulness.     

RFLP analysis for diagnosis of haemophilia A in the German Democratic Republic

The frequencies of Bcl I, Hind III and Xba I intragenic polymorphic sites in the population of the GDR were found to be 0.68, 0.38 and 0.48, respectively. No differences in composition and frequencies were detectable at DXS 52 locus in comparison with other Caucasian populations. A strong linkage disequilibrium between the intragenic Bcl I and Hind III sites could be confirmed. The observed heterozygosity for the flanking marker DXS 52 in combination with intragenic Bcl I and Xba I polymorphisms was 0.97. Using these three RFLPs, 122 females at risk in 41 independent haemophilia A families were investigated; 86 of them could be identified and 27 excluded as carriers; 9 females could not be classified. So far, four prenatal diagnoses in the first trimester of gestation have been performed by RFLP analysis.     

血友病B的基因诊断

血友病B是凝血因子Ⅸ基因突变引起血浆FⅨ量的缺乏或质的缺陷所导致的一种X连锁隐性遗传性出血性疾病,在男性中的发病率约为1/30 000,是一种严重危害人民健康的疾病.由于经济等方面的原因,我国血友病的防治与发达国家相比存在很大差距.目前尚缺乏针对本病的根治措施,故开展基因诊断及携带者检出无疑是防止新的患儿出生、阻断有害基因传递、提高人口素质的一个有效手段.     

First trimester prenatal diagnosis of haemophilia A using factor VIII gene probe

Accurate first-trimester prenatal diagnosis was achieved in a Japanese haemophilia A family by the use of a restriction fragment length polymorphism (RFLP) located within the F.VIII gene. Since the pregnant woman's heterozygosity for BclI polymorphism in F.VIII/intron 18 (F8A) probe was informative, chorionic villus sampling (CVS) was performed at 9 weeks of gestation. Restriction analysis showed that the fetus was heterozygous for the BclI site and had received a normal paternal X chromosome (0.9 kb) and a normal maternal X (1.2 kb). Therefore, we concluded that the fetus was a non-carrier female. Pregnancy went to term and woman gave birth to an apparently healthy female. At one week after birth a coagulation study confirmed that the newborn infant is not a carrier. The first-trimester prenatal diagnosis of haemophilia A is possible by CVS due to a RFLP in the F.VIII gene.     

Molecular diagnosis in hematopathology

The diagnosis of hematopoietic neoplasias has nowadays become more and more refined by molecular cytogenetic and molecular biological techniques. The biological basis of most of these techniques is that (malignant) tumors represent clonal neoplasias that are derived from a single or few progenitor cells, thus imparting a clonal relationship to all daughter cells. Apart from clonality analysis as a marker for neoplastic growth, the presence or absence of certain primary chromosome abnormalities, and the number and the kind of secondary genetic anomalies become increasingly important in the delineation of the biological grade of aggressiveness.     

Molecular diagnosis in allergy

Development and progress made in the field of recombinant allergens have allowed for the development of a new concept in allergy diagnosis, molecular diagnosis (MD), which makes it possible to identify potential disease‐eliciting molecules. Microarray‐based testing performed with a small amount of serum sample enables clinicians to determine specific‐IgE antibodies against multiple recombinants or purified natural allergen components. Performance characteristics of allergens so far tested are comparable with current diagnostic tests, but have to be confirmed in larger studies. The use of allergen components and the successful interpretation of test results in the clinic require some degree of knowledge about the basis of allergen components and their clinical implications. Allergen components can be classified by protein families based on their function and structure. This review provides a brief overview of basic information on allergen components, recombinants or purified, currently available or soon to become commercially available in ImmunoCAP or ISAC^® systems, including names, protein family and function. Special consideration is given to primary or species‐specific sensitization and possible cross‐reactivity, because one of the most important clinical utility of MD is its ability to reveal whether the sensitization is genuine in nature (primary, species‐specific) or if it is due to cross‐reactivity to proteins with similar protein structures, which may help to evaluate the risk of reaction on exposure to different allergen sources. MD can be a support tool for choosing the right treatment for the right patient with the right timing. Such information will eventually give clinicians the possibility to individualize the actions taken, including an advice on targeted allergen exposure reduction, selection of suitable allergens for specific immunotherapy, or the need to perform food challenges. Nevertheless, all in vitro tests should be evaluated together with the clinical history, because allergen sensitization does not necessarily imply clinical responsiveness. Cite this as: J. Sastre, Clinical & Experimental Allergy, 2010 (40) 1442–1460.     

Molecular diagnosis in breast cancer

Breast cancer is a complex and heterogeneous disease, encompassing a plethora of entities with distinct biological features and clinical behaviour. The advent of high throughput molecular methods has allowed a systematic characterization of the genomic landscape of breast cancer, revealing a profound heterogeneity in this disease. These methods are having a profound effect on the understanding of breast cancer. Some have already been incorporated in clinical practice, such as the prognostic ‘gene signatures’ that allow the tailoring of therapy in the subgroup of patients with oestrogen receptor (ER)-positive and HER2-negative breast cancer. In this review, we discuss the contribution of the main molecular methods in breast cancer research and how this information is changing our approaches to the diagnosis and management of this disease. We also address novel developments in the diagnosis and management of HER2-positive breast carcinomas and familial breast cancer.     

Molecular diagnosis in respiratory infections

Recent advance in molecular biology has enabled the specific and rapid diagnosis of the various infectious diseases. Though we commonly use the three major diagnostic procedure as isolation of the pathogen, direct detection of the pathogen and measurement of the immunological host reaction, DNA probe method would be the fourth major procedure in the clinical microbiology. The indication of the DNA probe method would be considered in the four cases as follows, 1. necessity of the special equipment to isolate the pathogen, 2. necessity of the long period to isolate the pathogen, 3. existence of the cross reaction among the pathogen and relative organisms in the immunological procedure, 4. existence of the difficulty to identify the species of the pathogen by the ordinary procedure. When we consider those indications, Legionnaires' disease might be one of the typical infectious disease to show the benefits of the DNA probe method in diagnosis. So far two types of DNA probe kits for Legionnaires' disease are available. One is the genus specific direct detection kit from the clinical specimens (Gen-probe), and the other is the microplate hybridization kit to identify each species of Legionella. The results of the evaluations of both kits showed the high specificity, rapidity and the clinical usefulness. In the next few years, various types of DNA probe kits might be newly developed and the contribution of those in the clinical microbiology would be much more than we expected.     

Molecular diagnosis in Huntington's disease

Huntington's disease (HD) is a neurological degenerative disorder, inherited by an autosomal dominant mode, and caused by a CAG triplet expansion coding for a poly-glutamine sequence in the huntingtin protein. HD affects 5-10 in 100,000 individuals from Caucasian population. Clinically patients display motor, cognitive and psychological impairment, and death within 10-15 years. Concrete advances have been achieved in the knowledge of the mutational mechanism, alteration of the protein product and their neuropathological effects. A number of tests such as PCR with or without DNA modification, Southern blot and mixed methods are analyzed. We describe their characteristics and effectiveness for the molecular diagnosis of HD.     

Molecular diagnosis in hematologic malignancies

Recent advances in molecular diagnosis in the hematological laboratory have greatly contributed to the diagnosis and treatment of hematologic malignancies, such as leukemia and lymphoma. The pathogenesis of leukemia and lymphoma has been disclosed by the analyses of genetic abnormalities in patients; abnormal gene expression may induce derangement in the control of cellular proliferation. Based on these genetic abnormalities, gene-targeted therapy has been introduced as a new approach to treating hematologic malignancies. We discuss here the usefulness of the molecular diagnosis in clinical hematology.     

High frequency of HLA-DR5 in Greek patients with haemophilia A and haemophilia B

Sixty unrelated Greek patients with haemophilia (46 with haemophilia A and 14 with haemophilia B) were typed for HLA-A, B and DR antigens. A highly significant increase in the frequency of HLA-DR5 was observed in both groups of patients (58.6% vs 30.0%, chi 2 = 10.47, pc less than 0.03, RR = 3.31 for haemophilia A and 78.5% vs 30.0%, chi 2 = 12.32, pc less than 0.007, RR = 8.5 for haemophilia B). An increased frequency of HLA-B13 was also observed in patients with haemophilia A (15.2% vs 5.7%, chi 2 = 5.74, pc less than 0.4, RR = 2.9). Thirty of the 60 patients (50.0%) were positive for LAV/HTLVIII antibodies. HLA-DR5 was equally distributed in patients with and without these antibodies (63.3% and 63.3%, respectively). The presence of DR5 did not correlate with the severity of haemophilia A or B. These results may suggest an influence of gene(s) on chromosome 6 in haemophilia A and haemophilia B and no effect of HLA antigens in the susceptibility to LAV/ HTLVIII infection among haemophiliac patients.     

Molecular diagnosis of influenza

The past decade has seen tremendous developments in molecular diagnostic techniques. In particular, the development of PCR technology has enabled rapid and sensitive viral diagnostic tests to influence patient management. Molecular methods used directly on clinical material have an important role to play in the diagnosis and surveillance of influenza viruses. Molecular diagnostic tests that allow timely and accurate detection of influenza are already implemented in many laboratories. The combination of automated purification of nucleic acids with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. The recent development of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to transform influenza diagnosis. While molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they remain invaluable in assisting our understanding of the epidemiology of influenza viruses.