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Query: UMLS:C0019045 (hemoglobinopathies)
 2,704 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper reviews the techniques presently available for the prenatal diagnosis of inherited hemoglobinopathies. At the present time, mutations of the globin genes are detected directly in trophoblast DNA, enzymatically amplified by polymerase chain reaction. Known mutations may be defined by restriction endonuclease digestion, non denaturing gradient gel electrophoresis, allele specific oligonucleotide probes or allele specific oligonucleotide primers. Unknown mutations are detected by denaturing gradient gel electrophoresis followed by direct sequencing. Other potentially useful methods for unknown mutations are single strand conformation polymorphism analysis and chemical mismatch cleavage analysis. A potential pitfall for all procedures based on analysis of amplified DNA is the coamplification of maternal sequences. This may be avoided by a careful dissection of maternal decidua from fetal trophoblast, by using an amount of chorionic villi not inferior to 5-10 mg and by reducing the number of amplifying cycles to approximately 20. Monitoring the presence of co-amplified maternal sequences by the analysis of polymorphic sequences is strongly recommended. Future perspectives consist of preimplantation diagnosis by biopsy of the morula or blastula or ova genotyping by analysis of the second polar body.
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PMID:Prenatal diagnosis of inherited hemoglobinopathies. 162 18

Restriction site polymorphisms are normal inherited variations in DNA that can be readily detected by restriction endonuclease analysis. Currently, 17 such polymorphisms are recognized within a 60 kb (kilobase) stretch of DNA which includes the beta-globin gene complex. Because of their proximity to the beta-globin gene, often these restriction site polymorphisms can be used to predict inheritance of beta-globin variants that produce disease. For example, restriction site polymorphisms can be used for prenatal diagnosis for the large majority of couples at risk of having a child with beta-thalassemia. When each member of such a couple is heterozygous at one or more of these 17 sites, family studies are usually successful in determining which forms of the polymorphism are co-inherited with the beta-thalassemia genes in that particular family. Subsequently, study of fetal DNA isolated from amniocytes obtained by midtrimester amniocentesis or from chorionic villi obtained by first trimester chorion biopsy will reveal which DNA polymorphisms that fetus has inherited. By deductive reasoning one can then predict which beta-globin genes it has co-inherited. Because of the general nature of these polymorphisms, which are related to the beta-globin gene and its variants only because of their proximity on chromosome 11, they are potentially useful in the prenatal diagnosis of any beta-chain hemoglobinopathy. Some hemoglobinopathies (including alpha-thalassemia, sickle cell anemia, and some cases of beta-thalassemia) can be detected directly by DNA analysis. In these cases in utero diagnosis does not need to rely on restriction site polymorphisms, which require preliminary family studies and are not applicable in all at risk pregnancies. Recently, genetic probes, which are necessary for detecting restriction site polymorphisms, have been isolated for sequences of several genes whose protein products are important in blood coagulation. These include probes for all three genes whose polypeptide products combine to form the fibrinogen molecule as well as probes for the prothrombin, Factor IX, Factor VIII, and antithrombin III genes. Defects in these genes are expected to be the causes of afibrinogenemia, prothrombin deficiency, hemophilia B, hemophilia A, and antithrombin III deficiency, respectively. From experience with other genes, it is expected that restriction site polymorphisms within and/or flanking these genes will be found.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Prenatal diagnosis of hemoglobinopathies by DNA analysis. 299 37

We report here an evaluation of 55 pregnancies at risk for a sickle hemoglobinopathy prenatally diagnosed by restriction-endonuclease analysis, with the endonucleases MstII and HpaI, of amniocyte DNA. The diagnosis was completed in all cases. Eleven fetuses were predicted to be affected, of which six were terminated. Forty-one of the 55 cases were confirmed. One false-negative was reported in a case predicted to be hemoglobin AS but that was determined to be hemoglobin SS at birth. We estimate that the 55 cases represent only 5% of the pregnancies at risk for a sickle hemoglobinopathy in the New York metropolitan area during the study period. We conclude that the prenatal diagnosis of sickle hemoglobinopathies by molecular methods is reliable. However, the efficiency of utilization and effectiveness of prenatal testing is dependent on the early prospective identification of couples at risk and on the education of communities concerning the significant morbidity of the sickle hemoglobinopathies and the reproductive choices now available to them.
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PMID:Prenatal diagnosis of sickle hemoglobinopathies: the experience of the Columbia University Comprehensive Center for Sickle Cell Disease. 303 20

We have summarized a number of different genetic disorders which can be diagnosed at the DNA level using restriction endonuclease fragment analysis. A whole spectrum of defects can be recognized: point mutations, deletions, additions, and crossing-over products or hybrid genes. These same restriction endonuclease techniques can enable different genes to be marked by polymorphism patterns. Thus, abnormal genes can be identified even if their exact DNA lesion is unknown or cannot be directly detected. The progress that has been made with the hemoglobinopathies and the experience from this group of single gene disorders should find application to other diseases as soon as specific probes become available.
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PMID:Genetic diseases: diagnosis by restriction endonuclease analysis. 628 49

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.
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PMID:Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy. 713 2

A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.
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PMID:Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette. 1252 64

A large number of mutations leading to hemoglobinopathies(abnormal hemoglobins, thalassemias) have been discovered. Gene diagnosis for a point mutation or a deletion/insertion of a few nucleotides is now readily performed. For a large deletion, once the precise breakpoints are unraveled, the same type of deletion is promptly diagnosed by gap PCR. However, a number of new types of large deletions remain unexamined. They need meticulous Southern blot analysis and/or cloning. Here we present a new technique to determine their precise breakpoints. One of the two breakpoints needs to be first assigned within the 5-kb portion estimated by gene dosage by quantitative PCR. The other breakpoint is left unexamined. The genome DNA is digested with one of eight kinds of endonucleases and subjected to recombination with pUC18 cloning vector digested with the same endonuclease. The gap PCR is subsequently performed between the common primers of pUC18 and five arbitrary primers within the aforementioned 5-kb portion. An abnormal gap PCR product, if detected by electrophoresis, discloses precise 5' and 3' breakpoints after direct sequencing. This method successfully disclosed the breakpoints for two epsilon gamma delta beta-thalassemias, one delta beta-thalassemia and one beta-thalassmeia in a relatively short period. All are new mutations. This method uses neither cloning procedures nor Southern blot, but employs gene dosage estimation and PCR. Thus, it is relatively simple. The progress of the genome project facilitated analysis of any large deletions.
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PMID:[Gene diagnosis of hemoglobinopathies--the determination of breakpoints for a large deletion]. 1288 42

beta-Globin gene cluster haplotypes were originally determined by restriction endonuclease mapping with Southern blots of polymorphic sites around the gene cluster. Over the years, haplotyping has been found to be useful, not only in population genetics but also in predicting the severity of hemoglobinopathies such as sickle cell disease. The sickle mutation occurs on five distinct haplotypes. The hitherto used methods are cumbersome and time-consuming, making haplotype determination a tedious procedure. We report our experience with a novel, rapid approach to haplotyping based on sequence polymorphisms in the Agamma-IVS-II region. We provide an algorithm that allows rapid assignment of the four African haplotypes carrying the sickle mutation.
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PMID:A novel approach to rapid determination of betaS-globin haplotypes: sequencing of the Agamma-IVS-II region. 1565 87