Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-29
2001-03-06
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S015000, C435S019000, C435S091200, C536S023200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06197507
ABSTRACT:
The present invention relates to &agr;-mannosidosis and its detection, in particular the detection of bovine &agr;-mannosidosis.
&agr;-mannosidosis is an autosomal, recessively inherited lysosomal storage disorder that has been clinically well characterised (M. A. Chester et al., 1982, in Genetic Errors of Glycoprotein Metabolism pp 90-119, Springer Verlag, Berlin). It is a very common disease in cattle but has also been found in man and cat. Glycoproteins are normally degraded stepwise in the lysosome and one of the steps, namely the cleavage of &agr;-linked mannose residues from the non-reducing end during the ordered degradation of N-linked glycoproteins is catalysed by the enzyme lysosomal &agr;-mannosidase (EC 3.2.1.24). However, in &agr;-mannosidosis, a deficiency of the enzyme &agr;-mannosidase results in the accumulation of mannose rich oligosaccharides. As a result, the lysosomes increase in size and swell, which impairs cell functions.
In man, the disease is rare. The symptoms of &agr;-mannosidosis include psychomotor retardation, ataxia, impaired hearing, vacuolized lymphocytes in the peripheral blood and skeletal changes. (Chester et al supra).
In cattle the disorder is much more common and results in mental retardation, skeletal changes, ataxia, a fine head tremor, aggressive behaviour and premature death. The disease has been reported among cattle in both Northern and Southern Hemispheres and in different breeds of cattle. Among cattle of the Angus breed there is a variation in phenotypic expression spanning from death within days to slowly progressing neurological deterioration that lasts for months (Healy et al., 1990, Res. Vet. Sci., 49: 82-84). Among other breeds of cattle such as Galloway, the symptoms can be more severe resulting in still birth or death within a few days after birth (Healy et al supra). As mentioned above, &agr;-mannosidosis is widespread in cattle and consequently is of considerable economic importance.
Currently, no treatment of the disease is available and consequently, not only are diseased cattle lost with respect to meat production, but it is important to prevent spread of the disease in breeding programmes. There is therefore a need to detect cattle that have or are carriers of &agr;-mannosidosis. A known method of detection has been to determine the enzyme activity of lysosomal &agr;-mannosidase. It is generally decreased to 40% of normal activity among cattle carrying &agr;-mannosidosis (ie. heterozygotes) and less than 10% in cattle exhibiting full &agr;-mannosidosis (ie. homozygotes). However, activity levels may vary between individuals and it sometimes becomes difficult to distinguish normal cattle from heterozygous carriers. This severely limits the utility of the enzyme test in detecting carriers.
The enzyme activity assay requires withdrawal of a blood sample less than 24 hours before analysis is begun. The enzyme activity is determined in isolated granulocytes (Healy, 1981, Res. Vet. Sci., 30: 281-283) so it is first necessary to isolate and purify the white blood cells. The enzyme activity is determined using the calorimetric substance p-nitrophenyl alpha D-mannopyranoside at pH 3.7 or 4-methyl umbelliferyl alpha D-mannopyranoside. This enzymatic test is very sensitive, contamination of the cells can result in erroneous results and in some instances it might be difficult to distinguish between heterozygous and normal values of &agr;-mannosidase activity.
The fact that only a short time can lapse between the taking of the sample from the animal and laboratory analysis means significant expense is incurred in ensuring speedy delivery to the laboratory and once at the laboratory samples have to be processed immediately, with the use of time consuming cell isolation techniques. The blood sample must usually be taken by a vet adding to the cost of the test. Thus, it will be seen that the disadvantages of the enzyme test are not inconsiderable and there remains a need for a straightforward and inexpensive text to detect cattle that have or are carriers of &agr;-mannosidosis.
The nucleotide sequence of human lysosomal &agr;-mannosidase has been determined (Nebes and Schmidt, 1994, Biochem. Biophys. Res. Comm., 200: 239-245) and a mutation causing &agr;-mannosidosis in humans identified, namely a base transition results in a His to Leu replacement in a conserved region of the gene for &agr;-mannosidase (Tollersrud et al., 1995, 10th ESGLD Workshop, Cambridge, England). However, in the case of cattle, full sequence information for the gene encoding bovine lysosomal &agr;-mannosidase (LAMAN) gene is not available, and more significantly, a corresponding mutation in the gene encoding LAMAN has not up to now been known. Consequently no gene based test for the disease in cattle has up to now been available.
Mutations in the gene encoding bovine LAMAN which cause &agr;-mannosidosis in cattle have now been elucidated, enabling and leading to the development of a genetic test for the disease.
Thus, in one aspect, the present invention provides a method for diagnosing or screening for bovine &agr;-mannosidosis, comprising detecting, in nucleic acid samples from cattle, the presence or absence of &agr;-mannosidosis-causing mutations in the gene encoding bovine LAMAN.
The present invention thus provides a method which not only enables the ready diagnosis of diseased cattle, but also permits cattle to be screened for the presence of the disease-causing allele of the LAMAN-encoding gene, enabling carriers to be detected and removed from breeding programmes. As used herein, the term “screening” thus includes detecting the presence of mutated LAMAN-encoding alleles in healthy cattle, ie. carriers, as well as in diseased animals. In this aspect, the invention can thus be seen to provide a genetic test for detecting the disease-causing &agr;-mannosidase gene in cattle.
As mentioned above, our studies have shown that point mutations in the LAMAN gene can be identified which are associated with the &agr;-mannosidase phenotype. Such &agr;-mannosidosis-causing mutations are thus base transitions in the LAMAN-encoding gene, leading to amino acid substitutions in the encoded LAMAN protein.
In a further aspect, the present invention thus provides a method of detecting &agr;-mannosidosis-causing mutations in cattle, comprising detecting the presence or absence of base transitions in the gene encoding bovine LAMAN, which are associated with disease.
As used herein the term “associated with disease” means that the base transitions result, as discussed above, in amino acid substitutions in the LAMAN protein which affect the functioning of the enzyme such that in homozygotes, &agr;-mannosidosis is caused.
As will be described in more detail in the Examples below, in work leading to the present invention, bovine LAMAN has been purified and been found to be encoded by a single gene. A cDNA encoding the bovine LAMAN protein has been prepared and sequenced (SEQ ID No. 1) and is shown in
FIG. 1
, together with its amino acid translation in single letter code. Genomic sequencing studies of the bovine LAMAN-encoding gene have also been undertaken and a partial genomic sequence is shown in
FIG. 2
(SEQ ID No. 3).
Such sequences represent further aspects of the invention. Thus, in a further aspect, the present invention can be seen to provide a nucleic acid molecule comprising all or a portion of a nucleotide sequence as shown in any one of
FIG. 1
or
2
(SEQ. ID Nos. 1 or 3) or a sequence which is complementary thereto, or which is a degenerate or allelic variant thereof, or a substantially homologous sequence having at least 85%, preferably at least 90% sequence identity therewith.
A still further aspect of the invention provides the use of a nucleic acid molecular comprising a nucleotide sequence as shown in
FIG. 1
or
2
or (SEQ ID Nos. 1 or 3) a sequence which is complementary thereto, or which is a degenerate or allelic variant thereof, or is a substantially homologous sequence having at least 85%, preferably at least 90% sequence identify therewith, or a
Berg Thomas
Nilssen Oivind
Tollersrud Ole Kristien
Johannsen Diana
Myers Carla J.
Rothwell Figg Ernst & Manbeck
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