Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
2001-07-18
2003-05-27
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C435S006120, C424S535000, C426S491000, C426S580000
Reexamination Certificate
active
06570060
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the use of milk which is free of the &bgr;-casein A
1
protein in the prevention or treatment of coronary heart disease. The invention also relates to the testing of DNA from cells obtained from lactating bovines for the presence of DNA encoding certain &bgr;-casein proteins, selecting the bovines on the basis of the testing, and then milking those bovines to produce milk free of &bgr;-casein A
1
for use in the prevention or treatment of coronary heart disease.
BACKGROUND OF THE INVENTION
Coronary heart disease is a major cause of death, particularly in countries where the populations are well-nourished, such as in the western world. Many factors are implicated as risk factors for this disease including obesity, smoking, genetic predisposition, diet, hypertension, and cholesterol.
Dairy products, especially milk, are a major contributor to the dietary intake of humans, again particularly in western world populations. Milk contains numerous components of nutritional and health benefit. Calcium is one example. However, milk is also a significant source of dietary fat. It is widely accepted that saturated fats found in milk are a risk factor for coronary heart disease. However, the inventor has discovered an additional risk factor present in some bovine milk unrelated to the fat content. What is entirely surprising is the source of the risk. The source is not dependent on the fat content of milk. Instead, it is a milk protein, &bgr;-casein, which is linked to coronary heart disease.
A number of variants of milk proteins have been identified. Initially, three variants of &bgr;-casein were discovered (Aschaffenburg, 1961) and were denoted A, B and C. It was later found that the A variant could be resolved into A
1
, A
2
and A
3
by gel electrophoresis (Peterson et. al. 1966). The &bgr;-casein variants now known are A
1
, A
2
, A
3
, B, C, D, E and F, with A
1
and A
2
being present in milk in the highest proportions. It is anticipated that other variants may be identified in the future.
The inventor has determined that it is the milk protein &bgr;-casein A
1
which represents the risk factor in bovine milk that is linked to coronary heart disease, or at least is the principal risk factor. This determination on the part of the inventor forms the basis of the present invention.
There is no relationship between the fat content of milk and &bgr;-casein genotype in cows. Therefore, selecting cattle on the basis of milk fat content will not identify which bovines produce the novel risk factor, namely the specific &bgr;-casein variant, in their milk.
There is no significant difference in the fat content of milk produced by cows which are homozygous for the &bgr;-casein A
1
allele (i.e. A
1
A
1
) and cows which are homozygous for the &bgr;-casein A
2
allele (i.e. A
2
A
2
). This is apparent from studies reported in the literature.
Ng-Kwai-Hang has carried out several studies. One study (Ng-Kwai-Hang et. al., 1990) suggested that milk containing &bgr;-casein A
1
rather than &bgr;-casein A
2
may have a slightly higher fat content. However, these differences were very small. The differences between milk from A
1
homozygous cows and milk from A
2
homozygous cows were 0.05% (for the first lactation period), 0.07% (for the second lactation period), and 0.04% (for the third lactation period).
In another study, Ng-Kwai-Hang (in an abstract cited by Jakob et. al;, 1990) found the opposite effect (i.e. the A
1
A
1
product had a lower fat content than the A
2
A
2
product). Thus, the 1995 Ng-Kwai-Hang abstract directly contradicts the Ng-Kwai-Hang, et. al., 1990 study.
McLean et. al., 1984 (McLean) also reported that there was no significant difference in the fat content of milk from cows of A
1
A
1
and A
2
A
2
genotypes (mean±standard error: 45.8±2.6 g/l for milk of A
1
A
1
cows and 48.6±1.9 g/l for milk of A
2
A
2
cows).
Aleandri et. al., 1990 (Aleandri), shows in Table 5 that the least squares estimates of the effects of different genotypes and their standard errors on fat percentage in milk are 0.12±0.09 for A
1
A
1
cows and 0.07±0.09 for A
2
A
2
cows. Taking into account the standard error for the test, Aleandri indicates that the effects of A
1
A
1
and A
2
A
2
genotypes on milk fat content are equivalent.
Bovenhuis et. al., 1992 (Bovenhuis), highlights that there are statistical problems associated with the way in which the genotype effects on fat percentages in milk are studied and documented. It is stated that the ordinary least squares estimates may be biased. Bovenhuis points out that the analysis of the effect of a particular genotype on various characteristics of milk is complex in nature and may, among other things, be affected by other genes which may be linked to the gene under study. Bovenhuis attempts to take into account the above variables and to overcome statistical problems by using an animal model method.
Table 3 of Bovenhuis indicates that, for a statistical model in which each milk protein gene is analysed separately and the A
1
A
1
cows designated as being the standard (i.e. given a value of 0% fat attributable to the genotype), the A
2
A
2
genotype was estimated not to contribute (i.e. 0%) to the fat content of the milk of the animals harbouring that genotype when compared to the A
1
A
1
genotype. The standard error of the test is recorded as 0.02%. Where a statistical model was used in which all milk protein genes were analysed simultaneously (Table 4 of Bovenhuis) and the A
1
A
1
genotype was again designated as being the standard (at 0% fat content attributable to the A
1
A
1
genotype), the A
2
A
2
genotype was estimated to contribute to the fat content of the milk at −0.01% when compared with the A
1
A
1
genotype. In this study a standard error of 0.02 was designated. Taking into account the standard error of the tests these results indicate that the A
2
A
2
genotype contributes to the fat content of milk in an equivalent manner to the genotype A
1
A
1
.
Gonyon et. al., 1987 reached the same conclusion as Bovenhuis.
The level of individual components in milk is influenced by both the genotype and the environment. That is, the variation between animals in milk output or milk composition is due to both genotypic and phenotypic factors. For example, Bassette et. al., 1988 (Bassette) indicates that the composition of bovine milk may be influenced by a number of environmental factors and conditions other than genetic factors. Environmental factors may impact on milk production and the constituents contained within the milk (including fat content). For example, changes in milk composition occur due to:
the stage of lactation (e.g., the fat content of colostrum is often higher; the concentration of fat changes over a period of many weeks as the cow goes through lactation);
the age of the cow and the number of previous lactations;
the nutrition of the cow including the type and composition of feed consumed by the cow;
seasonal variations;
the environmental temperature at which the cows are held;
variations due to the milking procedure (e.g., the fat content of milk tends to increase during the milking process which means that for an incomplete milking the fat content would generally be lower than normal and for a complete milking the fat content will be higher than normal); and
milking at different times of the day.
It is therefore apparent from the studies in this field that a person skilled in the relevant area of technology would not find a link between the fat content of milk and the &bgr;-casein genotype of the milk-producing bovines from which that milk is produced.
Thus, the inventor has for the first time identified the milk protein &bgr;-casein A
1
as a risk factor linked to coronary heart disease in its own right. It is therefore an object of this invention to provide a method of using milk substantially free of &bgr;-casein A
1
to prevent or treat coronary heart disease or to minimise the risk of developing coronary heart disease, or to at least provi
Crouch Deborah
Young & Thompson
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