Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-18
2001-04-24
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C435S201000
Reexamination Certificate
active
06221591
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of genotype analysis to determine the presence of a genetic risk factor with respect to susceptibility to infectious diseases in which pathogens having a chitin-containing coat are involved and other kinds of diseases, such as rheumatoid arthritis. This invention provides novel nucleic acid molecules which may be used in such genotype analysis and methods of genotype analysis using such novel nucleic acid molecules.
Furthermore, the subject invention is in the fields of diagnosis and detection of activated phagocytes in specific pathologies and changes therein during therapy.
BACKGROUND OF THE INVENTION
Gaucher disease is an inherited disorder characterized by the accumulation in tissues of glucosylceramide-laden macrophages (‘Gaucher cells’) as the result of a deficiency in the lysosomal enzyme glucocerebrosidase [1]. The presence of Gaucher cells is responsible for the common symptoms in Gaucher patients, such as hepatomegaly, splenomegaly, and skeletal deterioration [1].
The clinical manifestations in glucocerebrosidase-deficient individuals are very heterogeneous. Onset of clinical symptoms may occur at very young age, but the disorder may also remain virtually asymptomatic. Accurate prediction of disease severity and progression is not possible on the basis of the mutant glucocerebrosidase genotype of Gaucher patients.
During our search for sensitive markers for the presence of Gaucher cells, we discovered that in plasma of symptomatic patients chitotriosidase activity is markedly elevated [2]. Our present experience with more than 700 symptomatic Gaucher patients indicates that the average plasma chitotriosidase activity is about 1000-fold higher than the normal mean.
The discovery that activated storage macrophages are the source of the excessive plasma chitotriosidase in Gaucher patients, made us conceive that several disease conditions in which activated neutrophils and/or macrophages are involved might show detectable chitotriosidase abnormalities that can be exploited for diagnosis as well as for monitoring and optimalisation of therapeutic intervention. The feasibility and validity of this is illustrated by a number of examples, involving Gaucher disease, atherosclerosis, sarcoidosis, multiple sclerosis, arthritis and Crohn disease.
Copending U.S. patent application Ser. No. 08/486,839 describes our discovery that man contains a chitinase, named chitotriosidase, that fulfills a role in resistance against infections with chitin-containing pathogens by virtue of its ability to degrade chitin, an essential structural component of their coatings. Said patent application claims the use of chitotriosidase in therapy or prophylaxis against infectious diseases.
SUMMARY OF THE INVENTION
We herein describe the discovery of a novel genetic risk factor for infectious diseases, being an inherited abnormality in the chitotriosidase gene. A method that allows convenient analysis of chitotriosidase genotype is described.
Chitotriosidase genotype analysis is valuable in relation to prophylaxis of fungal infections and for the assessment of increased risk for other disease conditions such as (rheumatoid) arthritis. Information on the chitotriosidase genotype status also allows reliable interpretation of chitotriosidase enzyme activity levels.
Additionally, it is shown that chitotriosidase is a sensitive marker for monitoring activated phagocytes. It can be applied for diagnostic purposes as well as for monitoring efficacy of therapeutic intervention, in particular when also information about the chitotriosidase genotype of the examined individual is available. The value of chitotriosidase as marker for activated phagocytes is illustrated by a number of examples involving Gaucher disease, atherosclerosis, sarcoidosis, arthritis, multiple sclerosis, Crohn disease and neutrophil activation by G-CSF or GM-CSF administration.
Deficiency in chitotriosidase activity can be observed in specific individuals. About 5% of all subjects, including Gaucher patients, shows no true chitotriosidase activity due to the homozygous presence of a mutant chitotriosidase gene. About 35% of all individuals is carrier for this chitotriosidase defect. Chitotriosidase activity levels in materials (plasma, leukocytes, urine) of carriers are on average half those in control materials. Therefore, the interpretation of chitotriosidase activity levels without information on the chitotriosidase genotype of an individual is difficult. Consequently, there is a need for a convenient method that allows accurate identification of the chitotriosidase genotype.
Determination of the chitotriosidase genotype status of an individual is not only crucial for interpretation of enzyme activity levels in relation to diagnosis or monitoring of therapeutic intervention. Moreover, it is of great value for another reason. Infections with chitin-containing pathogens are a serious threat to mankind (for an extensive description see U.S. patent application Ser. No. 08/486,839). Most pathogens, with the exception of bacteria and viruses, have chitin as essential structural component in their coatings. It is known that chitinases in plants play an important role in defense against chitin-containing fungi [3]. Until recently it has generally been thought that vertebrates lack a similar defense mechanism. Our discovery that human phagocytes produce an analogous chitinase is therefore of importance.
Recombinant chitotriosidase has been found to be a potent inhibitor of hyphal growth of fungi like Candida and Aspergillus, and moreover it can protect mice against systemic infections with these pathogens. Systemic fungal infections are an increasing clinical problem for several reasons [4]. Firstly, the efficacy of the present treatment modalities is limited due to the development of resistance against antifungal agents and toxicity of the compounds. Secondly, the susceptibility for fungal infections is markedly increased when the immune system is suppressed. The number of individuals with a suppressed immune system increases (because of chemotherapy, transplantations, specific infections or genetic causes). Consequently, the incidence of systemic fungal infections, which are quite often lethal, has markedly increased in the last decade. There are indications that the genetic background may influence the susceptibility for infections. Most likely during immune suppression the resistance against pathogens heavily relies on innate defense mechanisms, of which chitotriosidase is one. It was conceived by us that a deficiency in chitotriosidase activity is an important risk factor with respect to susceptibility for chronic infection with and reduced resistance against chitin-containing pathogens, especially when the immune system is suppressed. About 5% of all subjects shows a deficiency in chitotriosidase activity, and is therefore at increased risk for particular infections. The elucidation of the genetic basis of inherited chitotriosidase deficiency allowed us to develop methods that sensitively detect carriers and homozygotes for the defect.
In conclusion, determination of the (hetero- or homo-allelic) presence of a 24 bp duplication in the chitotriosidase gene allows convenient identification of individuals that are at increased risk for developing a chronic infection with chitin-containing pathogens. Especially in the case of suppression of the immune system, a prophylactic treatment with antifungal agents (including chitotriosidase, see patent application Ser. No. 08/486,839) should be considered for those individuals carrying the chitotriosidase defect.
Determination of the chitotriosidase genotype is also valuable to assess the risk for other disease conditions such as (rheumatoid) arthritis. Information on the chitotriosidase genotype of an individual is moreover essential to interpret data on chitotriosidase activity levels in tissues and plasma that are for instance collected for diagnostic reasons or for the assessment of efficacy of
Hoffman & Baron LLP
Universiteit Van Amsterdam
Whisenant Ethan
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