Patent Application: US-62855403-A

Abstract:
a deletion mutant associated with ccr5 , particularly a ccr5 delta 32 deletion mutant , and its use for predicting the survival rate of patients having multiple sclerosis .

Description:
the presence in ms patients of the ccr5 delta 32 mutation , while not predisposing the patient to developing ms , was concluded to influence the course of the disease . 132 brain samples of deceased ms patients were examined for the ccr5 delta 32 mutation . results showed that the presence of ccr5 delta 32 made the patients more susceptible to an earlier death . specifically , observation of the 132 brain samples at autopsy showed that ccr5δ32 did not protect patients from developing ms when compared to controls , but contrary to expectations , made the ms patients more vulnerable to accelerated disease and earlier death . dna was isolated from post mortem brain tissue samples of 132 non - hispanic caucasians with ms and from whole blood of 163 healthy subjects . both groups were screened for the normal 1 allele and for the ccr5δ32 deletion 2 allele , using the polymerase chain reaction ( pcr ). alleles ( 1 and 2 ) and genotypes ( 11 , 12 and 22 ) were counted and their distributions between groups determined . survival analyses were used to test the effect of ccr5δ32 deletion on the survivorship . a significant association of the ccr5 mutant allele was found with early death and with a progressive reduction in the years of survival from age of onset to the age at death with increased frequency of the ccr5 deletion allele ( pl ≦ 0 . 00005 ). the death hazard ratio of ccr5 12 + 22 versus 11 genotype is 2 . 12 , after adjusting for the clinical subtype , suggesting ms patients with ccr5 genotype 12 or 22 have twice the mortality as compared to the 11 genotype of patients . interestingly , this effect is more significant in female ms patients than males , with the hazard ratio of 3 . 58 . ms subjects and controls were examined for differences in allelic frequencies and genotype prevalence . no significant variation was found among these groups . there was also no significant difference in age at onset of ms ( data not shown ). however , a marked difference was observed when the chi - square test was used to examine the potential progressive increase in certain genotypes of ms subjects across five groups with years of survival : ≦ 5 years , 6 - 10 years , 11 - 15 years , 15 - 20 years and ≧ 21 years ( see table 1 ). the ms subjects with 11 genotype survived progressively more years compared to heterozygous subjects who survived progressively fewer years ( p & lt ; 0 . 00001 ). additionally , these groups were examined for possible differences in mutant allelic frequencies by years of survival . a significant association of the ccr5 mutant allele ( 2 ) with an early death is shown in fig1 with a progressive reduction in the years of survival from age at onset to the age of death with increased frequency of the ccr5 deletion allele ( pl = 0 . 00005 ). the overall median survival time is 23 years ( range 1 - 56 ) after the onset of ms . comparing genotype 11 vs . 12 / 22 carriers , the median survival times are 24 and 16 years , respectively , while death rates are 0 . 039 and 0 . 056 for ms patients . fig2 shows kaplan - meier survival curves for patients with ccr5 genotypes . the cumulative death probability is significantly higher in the genotype 12 + 22 group than in the 11 group ( p ≦ 0 . 00099 ). for example , the cumulative death probabilities are 34 % and 69 % for the genotype group 11 and 12 + 22 after 20 years from onset . the 11 genotype was marginally significantly associated with the more benign clinical subtype ( p = 0 . 033 , data not shown ). because the survival difference can be confounded by clinical ms subtype and the age of onset , the cox regression model was used to test the main effect of ccr5 with simultaneous adjustment for these potential effects . the results showed that ms subtype was highly significant ( p ≦ 0 . 0001 ), while gender and age of onset were not . the hazard ratio was 2 . 12 for genotype 12 + 22 versus 11 with p value of ≦ 0 . 045 ( table 2a ) after adjusting for ms clinical subtype . this means that ms patients with ccr5 genotype 12 + 22 have over twice the mortality as compared to the 11 genotype , even after adjusting for the subtype . interestingly , this genotype effect mainly is seen in female patients ( table 2b ). the hazard ratio in females was 3 . 58 ( p - value ≦ 0 . 001 ; 95 % ci = 1 . 74 - 7 . 38 ). a potential bias may exist because those patients with unknown subtypes may have worse clinical diagnosis and shorter survival years . however , this effect more likely pushes estimates downward ( i . e . more conservative ) if these patients were associated with ccr5 genotype 12 . the above studies showed a significant gradient in survival for ms patients when individuals having mutant ccr5 genotypes were compared to those with wild type . thus , the presence of ccr5 mutation in patients with ms showed an exactly opposite effect compared to the protective effect seen in patients with hiv - 1 infection ( o &# 39 ; brien t r , et al ., 1997 ; deroda husman a - m , et al ., 1997 ). finally , the above results allow further speculation that an alteration in the immune response to stimulus ( viral perhaps ) occurs , whereby the response generated up - regulates rantes , and the immune response is directed away from the th1 to th2 cells . the effects of certain cytokines such as il - 10 and 13 will dominate over il - 2 , il - 12 and tnf - alpha . this polarization of t cells also alters their receptor expression and may be precipitated by the ccr5δ32 mutation . [ 0024 ] table 2a effect of ccr5δ32 on survivorship in ms patients by cox proportional hazard model , with adjustment of ms subtype ( n = 83 ) hazard ratio factors ( se ) z p - value lower 95 % upper 95 % onset age 1 . 03 ( 0 . 02 ) 2 . 00 0 . 045 1 . 00 1 . 06 ccr5 2 . 12 ( 0 . 56 ) 2 . 74 0 . 006 1 . 24 3 . 62 ms subtype 5 . 35 ( 1 . 44 ) 6 . 25 0 . 0001 3 . 16 9 . 06 [ 0025 ] table 2b effect of ccr5δ32 on survivorship in female ms patients by cox proportional hazard model , with adjustment of ms subtype ( n = 50 ) lower factors hazard ratio ( se ) z p - value 95 % upper 95 % ccr5 3 . 58 ( 1 . 32 ) 3 . 45 0 . 001 1 . 74 7 . 38 ms subtype 19 . 36 ( 10 . 24 ) 5 . 60 & lt ; 0 . 0001 6 . 86 54 . 60 the invention is further illustrated by the following examples , which are not intended to be limiting . dna was isolated from post - mortem brain tissue from 132 ms cases from the human brain and spinal fluid resource center at va los angeles health care center , los angeles , calif . all cases were necropsy - confirmed on the basis of white matter lesions and demyelination . the sample was comprised of 47 male and 85 female non - hispanic caucasians . the age distribution of ms cases had a range of 30 - 86 , with a mean of 57 . 2 ; the age at onset of ms ranged from 18 - 57 , with a mean onset age of 34 . 6 . ms subtyping was obtained by chart review for 83 of the subjects , while the remaining charts provided insufficient data for accurate subtyping . additionally , because a majority of the progressive forms are preceded by several years of relapsing - remitting course , and because the chart reviews relied more upon recent diagnostic information , the ms subtyping is probably biased toward more progressive forms . the control sample consisted of 163 adult college students from a nearby university ( 78 males and 85 females ), age range 18 - 49 , mean age 34 . 3 years , from whom blood samples were obtained for genetic studies . to detect ccr5 delta 32 mutations , genomic dna was extracted from brain samples and whole blood ( example 1 ) by standard procedures . a pcr based assay was used to determine the presence of the ccr5 delta 32 deletion . the following oligonucleotides were designed to yield a 232 bp product for the wild type : pcr was performed using an initial denaturation step at 94 ° c . for 4 minutes , followed by a second denaturation step at 94 ° c . for 45 minutes , an annealing step at 55 ° c . for 45 minutes , an elongation step at 72 ° c . for 45 minutes for 29 cycles , and a final elongation step at 72 ° c . for 6 minutes . pcr products were run on 12 % polyacrylamide gels at constant 150 v for 2 hours and stained with ethidium bromide and viewed on uv . the pcr product obtained had 232 bp for the wild type and a 32 bp deletion leading to 200 bp for the mutant allele . the data obtained on ccr5 delta 32 genotypes were subjected to statistical analysis . alleles and genotypes were counted and their distributions between groups were determined . the chi - square test was employed to statistically compare these groups . all statistical data calculations were done with the spss statistical package for macintosh ( release 6 . 1 . 1 ) ( spss , inc . ; chicago , ill .). survival analysis was applied to compare the survival time after ms onset between the ccr5 genotypes . to estimate the survivor function , kaplan - meier product - limit was used ( kaplan e l , et al ., 1958 ). log - rank test was used to examine the differences between two survivor curves of two ccr5 genotypes ( 11 versus 12 + 22 ). the cox proportional hazard model ( cox d r , et al ., 1984 ) was also applied to test the multivariate effects including ccr5 genotypic effect , gender and ms age of onset . these analyses were done in stata 7 . 0 ( stata corp ., college station , tex .). the above studies thus show a correlation between the ccr5 delta 32 deletion and early death by ms . in accordance with the present invention , the ccr5 delta 32 deletion may serve as a prognostic marker for ms . the publications and other materials used herein to illuminate the background of the invention , and to provide additional details respecting the practice of the invention , are incorporated herein by reference as if each was individually incorporated herein by reference . while the invention has been disclosed by reference to the details of preferred embodiments of the invention , it is to be understood that the disclosure is intended in an illustrative rather than a limiting sense , as it is contemplated that modifications will readily occur to those skilled in the art , within the spirit of the invention and the scope of the appended claims . 1 . karpus , w . j . et al . an important role of the chemokine macrophage inflammatory protein - 1 alpha in the pathogenesis of the t cell - mediated autoimmune disease experimental autoimmune encephalomyelitis . j immunol 1995 ; 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