Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-19
2001-03-13
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S810000, C536S027400, C536S028500, C536S029200
Reexamination Certificate
active
06200758
ABSTRACT:
FIELD OF THE INVENTION
The Inventor has developed and successfully applied a metabolic model of phenylalanine (Phe) metabolism to treatment development for neurological disorders, both to those seen only in psychiatric patients, and to those seen in the general population (U.S. Pat. No. 5,393,784; U.S. Pat. No. 5,670,539; allowed application U.S. application Ser. No. 08/545,095). The treatment method relates to a medical food product which has been clinically proven to reduce the availability of Phe to the central nervous system. The instant invention applies the Phe metabolism model to define and develop treatments to the genetics of psychotic, mood and personality disorders based on the results of (a) DNA analysis of the phenylalanine hydroxylase (PAH) gene, and (b) Phe dosing studies, in 124 patients with these disorders.
The PAH enzyme catalyzes the conversion of Phe to tyrosine (precursor of the amine neurotransmitter, dopamine), and deficient activity of this enzyme leads to increased plasma levels of Phe. Phe, as an indirect precursor of dopamine and noradrenaline, supports their synthesis at low plasma concentrations while at higher concentrations, it inhibits the synthesis of these two neurotransmitters in addition to serotonin. Thus plasma Phe levels play an important role in modulating neurotransmitter synthesis.
A model for Phe metabolism is valuable in the study of psychotic, mood and personality disorders (a) because of the role of Phe in amine neurotransmitter synthesis, (b) the suspected role of neurotransmitter function in these disorders, and (c) the fact that the limited treatment success that has been seen in these disorders is from agents that regulate neurotransmitter synthesis. Further, the acute sensitivity of the brain to higher than normal levels of Phe and its metabolites that occurs with deficiencies of PAH activity is well exemplified by the mental retardation, seizures, spasticity and EEG irregularities seen in phenylketonuria, a hyperphenylalanemia caused by deficient or absent PAH activity.
The psychotic disorders extract the greatest cost to the patient, family members, and to society at large. The most prevalent of the psychotic disorders both in the DNA study subject sample of the present invention and the general population is the disorder of schizophrenia. Schizophrenia is the most serious and the most treatment resistant of the disorders. It is heterogeneous in expression, etiology, and pathophysiology, with sex often being a defining variable. The disorder is known to have a complex pattern of inheritance, indicative of interactions among multiple genes and environmental factors and has been difficult to analyze at a genetic level. The present invention relates to methods of identifying and methods of treating the psychotic disorder of schizophrenia, though the invention is applicable to the entire group of psychotic, mood and personality disorders.
DNA analyses of the PAH gene in the study sample have revealed significant associations between the disorder of schizophrenia and both a novel missense mutation (K274E) and novel polymorphism (L321L). There was a statistical association between the presence of these two variants in the study sample, and all persons with either of these variants were of African-American ethnicity. The K274E missense mutation results in a lysine to glutamic acid substitution at amino acid 274, the substitution of which may cause changes in the physical and chemical properties of the PAH protein. The physiological effect of the mutation may be also be relevant to schizophrenia since the mutation is located in a region considered to be involved in the binding of tetrahydrobiopterin (BH
4
). BH
4
is the cofactor for Phe, tyrosine and tryptophan hydroxylases, all of which are required for the synthesis of the amine neurotransmitters dopamine, noradrenaline, and serotonin. These results suggest that the mutation may be relevant to the pathophysiology of schizophrenia.
Analysis of two-hour post Phe dose plasma Phe and tyrosine levels in the DNA study sample demonstrates (a) significantly higher Phe plasma levels, and (b) significantly less conversion to tyrosine, for those with the K274E mutation, indicating reduced catabolism of Phe for that group. In addition, differences in plasma levels for neopterin, biopterin and neopterin/biopterin ratio were observed between patients with and without the mutation, suggesting that increased synthesis of BH
4
may be associated with the K274E mutation. The increase in BH
4
synthesis may be a homeostatic response to reduced levels of PAH activity.
The effect of the novel L321L polymorphism on PAH enzyme activity is not known although the L321L polymorphism was found to be associated with schizophrenia and the K274E mutation. Studies of the variant may include investigations of DNA methylation and/or RNA splicing, stability or utilization. The significance of the L321L polymorphism is supported by the observation that patients with this polymorphism had significantly higher two-hour post Phe dose Phe plasma levels, and significantly less conversion to tyrosine, indicating reduced catabolism of Phe.
The nationally stated goal of the field of genetic research in psychotic, mood and personality disorders is to identify mutations that confer susceptibility to illness and that have predictable and understandable pathophysiological effects that may be related to these disorders. The present invention is a unique, hypothesis-driven approach toward meeting the goals of the field with (a) the discovery of a novel missense mutation and a novel polymorphism on a gene related to neurotransmitter synthesis in persons with psychotic, mood and personality disorders, (b) the discovery of a statistically significant association between the presence of these two variants, (c) the discovery of a statistically significant association between these two novel variants and the disorder of schizophrenia, (d) the discovery of a plasma dose response which supports significant physiological effects for the K274E mutation and L321L polymorphism and implicates these variants in the reduced catabolism of Phe, and (e) the discovery of a significant effect for the mutation in the increased synthesis of BH
4
. Further, the ethnic homogeneity for the two novel PAH variants associated with schizophrenia improves the prospects of making a meaningful contribution to the understanding the genetics of the complex disorder of schizophrenia. Ethnic considerations are of critical importance in genetic studies. For example, in patients with schizophrenia, significant differences were observed between persons of African and European descent with respect to heterozygosity and the number of alleles per marker, whereas in patients with Alzheimer's Disease, the APOE-e4 allele is consistently associated with a greater risk for the disease in Caucasians but not in African-Americans.
Treatment of the disorder of phenylketonuria is an example where early detection and therapeutic intervention has made a significant impact on the overall cost to society. The deleterious central nervous system effects of that disorder, also caused by mutations and polymorphisms on the PAH gene, have been significantly diminished in the western world because of newborn screening of Phe plasma levels. Infants with high levels of Phe are placed on a Phe-restricted diet, and as a direct consequence of such dietary restriction the central nervous system is protected from severe consequences of high plasma Phe such as mental retardation. The association of the novel K274E mutation and L321L polymorphism with schizophrenia in African-Americans in combination with pedigree analysis may also have enormous public health benefits with respect to the treatment of the disorder schizophrenia.
BACKGROUND OF THE INVENTION
The phenylalanine hydroxylase enzyme catalyzes the conversion of the large neutral amino acid phenylalanine (Phe) to tyrosine, which is the rate-limiting step in the catabolism of Phe. The brain is highly sensitive to levels of Phe, and def
Chakrabarti Arun Kv.
Fredman Jeffrey
New York State Office of Mental Health
Sughrue Mion Zinn Macpeak & Seas, PLLC
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