Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-16
2001-11-27
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091500, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06322981
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of diagnostic methodologies. More particularly, the invention is related to a simple, inexpensive and rapid method for detecting thalassemias and monitoring therapeutic effects on the disease.
BACKGROUND OF THE INVENTION
The &agr;-thalassemia (“&agr;-thal”) are common genetic disorders that result from reduced synthesis of the a globin chains of fetal (&agr;
2
&ggr;
2
, HbF) and adult (&agr;
2
&bgr;
2
, HbA) hemoglobin (Weatherall and Clegg, 1981; Higgs et al., 1989, Higgs and Weatherall, 1993). The normal human &agr; globin gene cluster is located on the short arm of chromosome 16 (Breuning et al., 1987; Buckle et al., 1988). In &agr;-thal syndromes, &agr;-globin synthesis is either diminished or absent due to either deletional or non-deletional abnormalities involving the &agr;-globin genes (Higgs et al., 1989; Higgs and Weatherall, 1993). Diploid cells have four &agr;-chain genes (i.e., &agr;&agr;/&agr;&agr;). The severity of the hematological and clinical picture is directly proportional to the number of involved &agr;-globin genes and thus the deletion of one, two, three, or all four of these &agr; genes attribute to mild to complete a chain deficiencies syndromes.
The deletion of &agr;-chain at birth results in the formation of a &ggr; chain tetramer, Hb Bart's (&ggr;
4
). The percentage of Hb Bart's present corresponds to the degree of &agr; chain deficiency (Cong & Shong, 1982; Liang et al., 1985; Higgs et al., 1989; Higgs and Weatherall, 1993). The &agr;-thal-2 genotype has been found to have one of the two genes deleted, thug these heterozygotes (&agr;&agr;/−&agr;) possess a mild &agr; chain deficiency resulting from the presence of only three &agr; chain genes and have not more than 2% Hb Bart's at birth (Higgs et al., 1989). Homozygotes (−&agr;/−&agr;), as well as &agr;-thal-1 heterozygotes (&agr;&agr;/−−), possess moderate a chain deficiency resulting from the presence of only two &agr; chain genes and have approximately 5% Hb Bart's at birth (Higgs and Weatherall, 1993). While Hb H disease is a heterozygosity for &agr;-thal-2 in association with an &agr;-thal-1 heterozygosity (−−/−&agr;), a severe &agr; chain deficiency occurs due to the deletion of three &agr; chain genes (Higgs et al., 1989; Higgs and Weatherall, 1993). The &agr;-thal-1 homozygotes (−−/−−), which lack any functional &agr; genes, present with Hb Bart's is known as hydrops fetalis syndrome and results in intra-uterine or early post-delivery death to the fetus (Lie-Injo & Hie, 1960; Higgs et al., 1989). Mother bearing fetuses with homozygous &agr;-thal-1 (−−/−−) are at high risk for obstetrical complications, such pregnancy induced hypertension, eclampsia, and/or death.
At present, for a variety of technical, logistical and economical reasons, large-scale carrier screening and appropriate ante-natal detecting programs have not been established for the populations in which this syndrome occurs (Bowden et al., 1992; Higgs and Weatherall, 1993).
It is an object of the invention to develop a diagnostic method and kit for the detection and quantitation of hemoglobin (Hb) &agr; gene(s) in &agr;-thalassemia patients.
It is another object of the present invention to develop a method and kit for screening for carriers of the genetic disorder, &agr; thalassemia.
It is yet another object of the present invention to identify persons who are at risk of having offspring with homozygous &agr;-thalassemia as well as identify &agr;-thalassemia in individuals with unexplained microcytosis and hypochromia.
Another object of the present invention is to develop a sensitive, non-radioisotopic test capable of differentiating between the various forms of &agr;-thalassemia, by detecting and quantitating a gene(s) from blood samples.
SUMMARY OF THE INVENTION
The present invention relates to the identification of primers capable of detecting and distinguishing between the various forms of &agr;-thalassemia. The &agr;-globin primers of the present invention are derived from the &agr;-globin genes. One such primer comprises a region common to both &agr;
1
and &agr;
2
genes. A second such primer is specific for &agr;
2
gene and a third primer is specific for &agr;
1
gene. These primers are designed to specifically hybridize to portions of the hemoglobin &agr; genes, such that, when amplified with an inducing agent, give identifiable amplification products. The presence and amount of the products is correlated to specific forms of &agr;-thalassemia, based upon the type of gene deletion associated with the disease.
The present invention relates to a method for identifying and differentiating among the various forms of &agr;-thalassemia. The method of the present invention uses highly specific primers to identify and differentiate among the forms of &agr;-thalassemia. The assay may include a nucleic acid amplification step wherein the hemoglobin a genes are simultaneously amplified and detected. In another embodiment of the present invention, a color complementation assay is used to provide a convenient, rapid, non-radioisotopic, assay system for quantitation of the &agr; gene(s).
A kit comprising at least three primers, capable of detecting, quantitating and distinguishing &agr;-thalassemia variants is also encompassed in the present invention.
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patent: 97 16568 (1997-05-01), None
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Bowie, L.J. et al. (1994) “Detection of &agr;-Thalassemias by multiplex polymerase chain reaction”Clin. Chem.40 (12):2260-2266.
Wen, X-J. et al., (1992) “The nondeletional types of HB H disease in Guangxi”Hemoglobin16(1&2): 45-50.
Liu, T-C. et al. (1994) “Molecular basis and hematological characterization of Hb G disease in southeast asia”Am. J. of Hematology45: 293-297.
Orkin, S.H. et al. (1981) “Mutation in an intervening sequence splice junction in man” Proc. Natl. Acad. Sci. USA 78 (8): 5041-5405.
Bowden, D.K. et al. (1992) “A PCR-based strategy to detect the common severe determinants of &agr; thalassaemia” Brit. J. Haematolgy 81: 104-108.
Rodgers Griffin P.
Tang Delia C.
Auth Dorothy R.
Feiler William S.
Johannsen Diana
Morgan & Finnegan L.L.P.
Myers Carla J.
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