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A human UDP galactose-4-epimerase polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the treatment of galactosemia. Also disclosed are diagnostic assays for the detection of mutations in the nucleic acid sequencing encoding human UDP galactose-4-epimerase protein and for detecting an altered level of human UDP galactose-4-epimerase protein in a sample derived from a host.

Inventors: Hongjun Ji, Craig A. Rosen
Original Assignee: Human Genome Sciences, Inc.


Carbohydrates are major sources of energy in animals, plants and many microorganisms. They can also be precursors in the biosynthesis of other compounds, such as fatty acids, triglycerides, and some amino acids. Carbohydrates are also important as structural components of connective tissue, nervous tissue, bacterial cell walls, and nucleic acids.

Disaccharides ingested by higher animals are usually hydrolyzed to their monosaccharide components before absorption into the intestine. For example, sucrose is converted into glucose and fructose, maltose is converted into glucose, and lactose is converted into glucose and galactose in most cases by enzymatic activity. It is these monosaccharides that are eventually used for the biosynthesis of carbohydrates. The L forms of the monosaccharides are of small consequence and it is the D form which are most often utilized in the biosynthesis of carbohydrates.

Galactose is an important precursor in carbohydrate biosynthesis and the synthesis of many other macromolecules which are vital to the normal functioning of a mammalian system.

Uridine diphosphate-galactose (UDP-galactose)is also an important intermediate in the metabolism of free D-galactose, formed by the enzymatic hydrolysis of lactose or milk sugar in the intestinal tract. D-galactose is converted into D-glucose in the liver by two series of reactions which have attracted much attention because they are subject to genetic defects in man, resulting in different forms of the hereditary disease galactosemia. Galactosemia results from three known forms of deficiency, galactokinase deficiency, galactose-1-phosphate uridyltransferase deficiency and UDP-galactose-4-epimerase deficiency.

In the liver, free D-galactose is first phosphorylated at carbon atom 1 by galactokinase, to yield D-galactose-1-phosphate, which is converted into UDP-galactose by one of two possible reactions. The minor route is catalyzed by UTP galactose-1-phosphate uridyltransferase.

The UDP-galactose so formed is normally converted into UDP glucose by UDP-galactose-4-epimerase. These reactions make it possible for the galactose residue to enter into the main pathways of glucose metabolism, since UDP-glucose, as we shall see, can donate its glucose residue to glycogen. This uridyltransferase is present in high amounts in the liver of adults but lacking in infants.

The second pathway for utilization of free galactose also begins with galactose 1-phosphate. It is catalyzed by UDP-glucose: galactose 1-phosphate uridyltransferase. This particular enzyme is present in normal fetal liver but is lacking in infants with one form of galactosemia. Such infants are thus unable to utilize galactose by either pathway. Galactosemic infants have an excessively high concentration of D-galactose in the blood and suffer from cataracts of the lens of the eye, mental disorders, impairment of the peripheral nervous system, blindness, hearing deficiency, organomegaly, deranged liver function, including elevated blood galactose, galactosuria, hyper-cholbremic acidosis, albuminuria and aminoaciduria (Mason, H. H. and Turner, M. E., Am. J. Dis. Child, 50:359 (1935) and Komrower, G. M. et al., Arch. Dis. Child, 31:254 (1956)).

Mental retardation is the most significant outcome of galactosemia. The extent of retardation is characterized in that extremely low IQ values result. Psychological problems also seem to be prevalent, with inadequate drive, shyness and withdrawal (Nadler, H. L., et al., Galactosemia, Springfield, Ill., Charles C. Thomas, p. 127 (1969) and Komrower, G. M. and Lee D. H., Arch. Dis. Child, 45:367 (1970)). Other results include a high incidence of ovarian failure with hypergonadotrophic hypogonadism in females who have had adequate dietary therapy (Kaufman, F. R. et al., N. Engl. J. Med., 305:994 (1981); Steinmann, B. et al., N. Engl. J. Med., 305:464 (1981); Kaufman, F. R., et al., J. Inherited Metab. Dis., 9:140 (1986); Robinson, A. C. R., et al., Br. J. Obstet. Gynaecol., 91:199 (1984); and Fraser, I. S., et al., Clin. Reprod. Fertil., 4:133 (1986)). These deficiencies are only a few of the deficiencies which result from this disease and there are numerous other disorders and anatomical and biological problems which result from this disease. The second type of galactosemia is similar to the first type with minor variations in the biological causes and resulting diseases.

Uridine diphosphate galactose-4-epimerase is the third enzyme in the metabolism of dietary galactose and the key enzyme in de novo synthesis of galactose metabolites from glucose (Gitzelmann, R., and Steinmann, B., Enzyme, 32:37-46, (1984)). UDP galactose-4-epimerase catalyzes a reversible transaction between UDP-glucose and UDP-galactose, and a deficiency of this enzyme results in galactosemia.

The first case of epimerase deficiency was documented in 1972 by Gitzelmann in the blood cells of an apparently healthy infant (Gitzelmann, R., Helv. Paediat. Acta, 27:125-130 (1972)). Subsequent clinical findings indicated that severe clinical manifestations of galactosemia were usually observed in patients with generalized epimerase deficiency and were indistinguishable from the classic galactosemia caused by galactose-1-phosphate uridyltransferase deficiency (Henderson, M. J., and Holton, J. B., J. Inher. Metab. Dis., 6:17-20 (1983)). The incidence of complete absence of epimerase activity in circulating blood cells in estimated to be 1 in 23,000 in Japan (Misumi, H. et al., Clinica Chimica Acta, 116:101-105 (1981)). Somatic cell hybrid studies have defined an area from chromosome 1 pter-p21 that likely contains a gene for galactose epimerase (Benn, P. A. et al., Cytogenet. Cell Genet., 24:138-142 (1979) and Lin, M. S. et al., Cytogenet. Cell Genet., 24:217-223 (1979)).

Galactosemia in general is inherited as an autosomal recessive trait. Also, the enzyme may be present, but non-functional due to a mutation in the gene sequence encoding the functional enzyme.


The gene of the present invention has been putatively identified as a human UDP-galactose-4-epimerase. This identification has been made as a result of amino acid sequence homology to the rat galE MRNA for UDP-galactose-4-epimerase.

In accordance with one aspect of the present invention, there are provided novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptide of the present invention, including mRNAs, DNAS, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with yet a further aspect of the present invention, there are provided processes for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding the polypeptide of the present invention under conditions promoting expression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, to treat galactosemia, cataracts, mental disorders, impairment of the peripheral nervous system, blindness, hearing deficiency and organomegaly.

In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polypeptide of the present invention.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

This invention also provides a method to diagnose human UDP galactose-4-epimerase deficiency which comprises isolating a nucleic acid sample from an individual and assaying the sequence of said nucleic acid sample with the referenced gene of the invention and comparing differences between said sample and the nucleic acid of the instant invention, wherein said differences indicate mutations in the human UDP galactose-4-epimerase gene isolated from an individual.

This invention also provides a method for treating conditions which are related to insufficient human UDP galactose-4-epimerase activity via gene therapy. An additional reference gene comprising the UDP galactose-4-epimerase gene of the instant invention is inserted into a patient's cells either in vivo or ex vivo. The referenced gene is expressed in transfected cells and as a result, the protein encoded by the referenced gene corrects the defect thus permitting the transfected cells to function normally and alleviate disease conditions (or symptoms).

In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA, manufacture of DNA vectors and the treatment and diagnosis of human disease.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.