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Analyses of a MS-associated haplotype encompassing the CCL3 gene
Journal of Neuroimmunology 176 (2006) 216 – 218 www.elsevier.com/locate/jneuroim
Short communication
Analyses of a MS-associated haplotype encompassing the CCL3 gene Tamara Vyshkina, Bernadette Kalman * Department of Neurology, Saint Luke’s Roosevelt Hospital Center, Columbia University, 432W 58th Street, Room 226, New York, NY 10019, United States Received 23 November 2005; received in revised form 21 March 2006; accepted 27 March 2006
Abstract Our previous studies showed the association of multiple sclerosis with the same marker haplotype encompassing the CCL3 gene in two independent sets of families. Here we present that sequencing of this haplotype and its flanking regions detected no new mutation, but 16 single nucleotide polymorphisms (SNP) and 1 insertion/deletion variant in both affected and unaffected individuals. Transmission distortion analyses of the newly identified variants in the second set of families revealed no individual marker association. In the absence of a single disease relevant variant within the MS associated haplotype and the surrounding linkage disequilibrium block, the highlighted haplotype may itself indicate a functionally relevant allelic combination or interaction. D 2006 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; LD mapping; Genetic association; CCL3
1. Introduction The involvement of CC chemokine ligands (CCL) and their receptors (CCR) in the development of multiple sclerosis (MS) is well established, but without a direct link between genetic variations and immunopathological observations (The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001; Mahad et al., 2004; Kantarci et al., 2005; Banisor et al., 2005). One of the most consistent immunological observations implicates CCL3 and CCL5 in the recruitment of mononuclear cells expressing CCR1 and CCR5 receptors into active lesions of MS. While CCL3 and CCL5 are present in acute and chronic active plaques, and increased levels of these chemokines are detected in the cerebrospinal fluid (CSF) of patients during relapses, the expression of CCR1 and CCR5 also can be demonstrated on lymphocytes, monocytes and macrophages in blood, CSF and chronic active plaques of MS patients (Banisor et al., 2005). CCR1+/CCR5+ monocytes are present in all four types of early, actively demyelinating plaques. However, monocytes and macrophages lose their CCR1 positivity, and express the CCR1 /CCR5+ pheno* Corresponding author. Tel.: +1 212 523 8676; fax: +1 212 523 8859. E-mail address: [email protected] (B. Kalman). 0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.03.018
type similar to that of microglia in late active type II but not in type III lesions (Lucchinetti et al., 2000; Mahad et al., 2004). These observations suggest the involvement of CCL3– CCR1/CCR5 and CCL5 –CCR1/CCR5 ligand-receptor interactions in the development of MS, but with distinct temporal characteristics in pattern II and III lesions. Genetic studies also support the involvement of CCLs in MS. A meta-analysis of raw genotype data from three full genome scans indicates the highest score of non-parametric linkage (NPL) = 2.58 at 17q11 encoding a cluster of 14 CCLs along with other candidate genes of MS (The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001). To further confine the region of interest, we performed single nucleotide polymorphism (SNP) genotyping and family based association studies within the 1.85 MB CCL cluster. First, testing the transmission distortion of SNP marker alleles in 257 Caucasian families, we identified MS associated haplotypes encompassing the CCL2, CCL11 –CCL8– CCL13, CCL15 and CCL3 genes (Vyshkina et al., 2005). In a second association study with 232 markers in the same region (average pacing within the entire region <8 kb, pacing within CCL genes < 1 kb, pacing within CCL3 < 0.6 kb) in an independent set of 361 families, we confirmed the existence of MS associated haplotypes
T. Vyshkina, B. Kalman / Journal of Neuroimmunology 176 (2006) 216 – 218
defined by the same markers within CCL3, partly identical markers within CCL15 and co-localizing but different markers between CCL2 and CCL7 (Vyshkina and Kalman, 2005). In order to determine if new mutations are present within or in the proximity of a MS-associated haplotype, we chose the best defined haplotype encompassing CCL3 for sequence analysis.
2. Material and methods 2.1. Patients and families Altogether, 17 affected and 8 unaffected unrelated individuals were identified with the MS associated threemarker haplotype encompassing CCL3 in 1369 individuals from 361 trio, affected sibpair, incomplete and multiplex Caucasian families (details in Vyshkina and Kalman, 2005). All positive individuals were heterozygous for this haplotype defined by the A – T – C alleles of SNPs 278 –277 –273. 2.2. Automatic sequencing A 3500-kb DNA segment, including the MS-associated three-marker haplotype (1180 bp) and its 5V(1750 bp) and 3V (570 bp) flanking regions, was amplified by PCR in 7 overlapping fragments. An additional pair of primers was designed to avoid the interference of an insertion/deletion [between nt1487 and nt1495] with the annealing of the colocalizing forward primer in PCR segment 5 in some individuals. The DNA fragments were directly sequenced on an ABI Prism 3700 DNA Analyzer (Genaissance Pharmaceuticals, Inc., New Haven, CT). Sequences were aligned with the corresponding segment of the NC_010799 contig on chromosome 17 (http://www.ncbi.nlm.nih.gov/ entrez/viewer.fcgi?val=37544509&view=gbwithparts). 2.3. Genotyping SNPs revealed by sequencing were genotyped using the Sequenom MassARRAY System in the second set of 361 families. Associations of these alleles and haplotypes with MS were tested by using the pedigree disequilibrium test
217
(PDT), Family Based Association test (FBAT), the TRANSMIT version 2.5 program and Haplotype Based Association Test (HBAT) as previously described (Vyshkina and Kalman, 2005).
3. Results and discussion In search for a MS-related mutation, we sequenced an extended MS-associated haplotype defined by SNPs 278– 277 – 273 encompassing the CCL3 gene. This haplotype was only found in heterozygous state in 17 affected and 8 unaffected individuals out of 1369 in 361 families (Vyshkina and Kalman, 2005). 5Vand 3Vto the core haplotype within the extended region subjected to sequencing, there were two additional SNPs (280 and 271) that showed no direct association with the disease in the previous family studies (Fig. 1) (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). Similarly, none of the densely placed markers 5V or 3V to the selected 3500 bp DNA segment within the chromosomal block of linkage disequilibrium (LD) revealed association with MS in either of the previous studies (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). Therefore, we postulated that if a MS-relevant mutation is present, it must be within or in the proximity of the haplotype defined by SNPs 278 – 277– 273. Nevertheless, sequencing of the selected region revealed no new mutation, but 16 SNPs and 2 insertion/deletion variants in the 25 affected and unaffected individuals with the haplotype. All but two individual variants within the MS-associated chromosomes can be found in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/). Six of 16 SNPs were included but did not show association with MS in the previous family studies (Vyshkina and Kalman, 2005), and one insertion/deletion appeared only in the non-MS-associated chromosomes. Thus, we excluded these 9 variants from further genotyping and association analyses. The remaining variants were genotyped in 1369 individuals, in 361 families (described in Vyshkina and Kalman, 2005). The allele distribution of these markers was in agreement with the Hardy-Weinberg equilibrium, and none of them showed association with the disease using PDT and FBAT. In the TRANSMIT and HBAT analyses of pair-wise two-marker
Fig. 1. The sequenced 3500 base pair region with all variations revealed. The horizontal bar represents the sequenced 3500 bp DNA segment including the MS associated haplotype and its flanking regions. Introns, exons and the 5Vand 3VUTRs of CCL3 are indicated. The 16 SNPs and one insertion/deletion variant detected within the MS associated chromosomes in 25 individuals are designated by dots within the DNA symbol. Black dots represent SNPs 280, 278, 277, 274, 273 and 271 involved in previous genotyping and association studies. The three larger dots represent SNPs 278, 277 and 273 defining the MS associated haplotype. The SNP between markers 278 and 277 had a minor allele frequency of 0.002, and thus was not informative in the analyses.
218
T. Vyshkina, B. Kalman / Journal of Neuroimmunology 176 (2006) 216 – 218
haplotypes, an allelic combination of markers encompassing the 3VUTR of CCL3 and its 3V flanking region showed association in the HBAT ( p = 0.012), but this association was weak and the haplotype fell within the 3V segment of haplotype 278 – 277 – 273 (for which, a TRANSMIT p = 0.0023 and a HBAT p = 0.0078 was previously detected in the same set of families) (Vyshkina and Kalman, 2005). After completing the association analyses of all variants and haplotypes in this region, the strongest and most consistently observed finding remained the association with the A – T allelic combination of SNPs 278– 277 (TRANSMIT p = 0.0001, HBAT p = 0.0045) in the exon II and intron I of CCL3 (Fig. 1). In summary, sequence analyses of an MS associated haplotype and its flanking regions encompassing the gene of CCL3 revealed no new mutation. The detected sequence variations also did not show association with the disease when genotyped and tested for transmission distortion in families. Although the existence of a disease relevant variant in LD with the investigated extended haplotype may be postulated, it is not likely given the lack of SNP or haplotype associations in two independent studies with dense marker pacing in the surrounding LD block (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). After these sequential and comprehensive analyses, the strongest positive finding remains the association of haplotype 278A – 277T with MS in the CCL3 gene. Taken into consideration the established role of CCL3 in plaque development and the exclusion of other genetic markers, this haplotype itself may indicate a functionally important allelic interaction in MS.
Acknowledgments This study was supported by the RG 3521-A-6 grant from the National Multiple Sclerosis Society.
References Banisor, I., Leist, T.P., Kalman, B., 2005. Involvement of h-chemokines in the development of inflammatory demyelination. J. Neuroinflamm. 2, 7 (Electronic publication). Kantarci, O.H., Morales, Y., Ziemer, P.A., Hebrink, D.D., Mahad, D.J., Atkinson, E.J., Achenbach, S.J., De Andrade, M., Mack, M., Ransohoff, R.M., Lassmann, H., Bruck, W., Weinshenker, B.G., Lucchinetti, C.F., 2005. CCR5Delta32 polymorphism effects on CCR5 expression, patterns of immunopathology and disease course in multiple sclerosis. J. Neuroimmunol. 169, 137 – 143. Lucchinetti, C., Bruck, W., Parisi, J., Scheithauer, B., Rodriguez, M., Lassmann, H., 2000. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707 – 717. Mahad, D.J., Trebst, C., Kivisakk, P., Staugaitis, S.M., Tucky, B., Wei, T., Lucchinetti, C.F., Lassmann, H., Ransohoff, R.M., 2004. Expression of chemokine receptors CCR1 and CCR5 reflects differential activation of mononuclear phagocytes in pattern II and pattern III multiple sclerosis lesions. J. Neuropathol. Exp. Neurol. 63, 262 – 273. The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001. A metaanalysis of genomic screens in multiple sclerosis. Mult. Scler. 7, 3 – 11. Vyshkina, T., Kalman, B., 2005. Haplotypes within genes of h-chemokines are associated with multiple sclerosis: a second phase study. Hum. Genet. 118, 67 – 75. Vyshkina, T., Shugart, Y.Y., Birnbaum, G., Leist, T.P., Kalman, B., 2005. Association of haplotypes in the beta-chemokine locus with multiple sclerosis. Eur. J. Hum. Genet. 13, 240 – 247.
Short communication
Analyses of a MS-associated haplotype encompassing the CCL3 gene Tamara Vyshkina, Bernadette Kalman * Department of Neurology, Saint Luke’s Roosevelt Hospital Center, Columbia University, 432W 58th Street, Room 226, New York, NY 10019, United States Received 23 November 2005; received in revised form 21 March 2006; accepted 27 March 2006
Abstract Our previous studies showed the association of multiple sclerosis with the same marker haplotype encompassing the CCL3 gene in two independent sets of families. Here we present that sequencing of this haplotype and its flanking regions detected no new mutation, but 16 single nucleotide polymorphisms (SNP) and 1 insertion/deletion variant in both affected and unaffected individuals. Transmission distortion analyses of the newly identified variants in the second set of families revealed no individual marker association. In the absence of a single disease relevant variant within the MS associated haplotype and the surrounding linkage disequilibrium block, the highlighted haplotype may itself indicate a functionally relevant allelic combination or interaction. D 2006 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; LD mapping; Genetic association; CCL3
1. Introduction The involvement of CC chemokine ligands (CCL) and their receptors (CCR) in the development of multiple sclerosis (MS) is well established, but without a direct link between genetic variations and immunopathological observations (The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001; Mahad et al., 2004; Kantarci et al., 2005; Banisor et al., 2005). One of the most consistent immunological observations implicates CCL3 and CCL5 in the recruitment of mononuclear cells expressing CCR1 and CCR5 receptors into active lesions of MS. While CCL3 and CCL5 are present in acute and chronic active plaques, and increased levels of these chemokines are detected in the cerebrospinal fluid (CSF) of patients during relapses, the expression of CCR1 and CCR5 also can be demonstrated on lymphocytes, monocytes and macrophages in blood, CSF and chronic active plaques of MS patients (Banisor et al., 2005). CCR1+/CCR5+ monocytes are present in all four types of early, actively demyelinating plaques. However, monocytes and macrophages lose their CCR1 positivity, and express the CCR1 /CCR5+ pheno* Corresponding author. Tel.: +1 212 523 8676; fax: +1 212 523 8859. E-mail address: [email protected] (B. Kalman). 0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.03.018
type similar to that of microglia in late active type II but not in type III lesions (Lucchinetti et al., 2000; Mahad et al., 2004). These observations suggest the involvement of CCL3– CCR1/CCR5 and CCL5 –CCR1/CCR5 ligand-receptor interactions in the development of MS, but with distinct temporal characteristics in pattern II and III lesions. Genetic studies also support the involvement of CCLs in MS. A meta-analysis of raw genotype data from three full genome scans indicates the highest score of non-parametric linkage (NPL) = 2.58 at 17q11 encoding a cluster of 14 CCLs along with other candidate genes of MS (The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001). To further confine the region of interest, we performed single nucleotide polymorphism (SNP) genotyping and family based association studies within the 1.85 MB CCL cluster. First, testing the transmission distortion of SNP marker alleles in 257 Caucasian families, we identified MS associated haplotypes encompassing the CCL2, CCL11 –CCL8– CCL13, CCL15 and CCL3 genes (Vyshkina et al., 2005). In a second association study with 232 markers in the same region (average pacing within the entire region <8 kb, pacing within CCL genes < 1 kb, pacing within CCL3 < 0.6 kb) in an independent set of 361 families, we confirmed the existence of MS associated haplotypes
T. Vyshkina, B. Kalman / Journal of Neuroimmunology 176 (2006) 216 – 218
defined by the same markers within CCL3, partly identical markers within CCL15 and co-localizing but different markers between CCL2 and CCL7 (Vyshkina and Kalman, 2005). In order to determine if new mutations are present within or in the proximity of a MS-associated haplotype, we chose the best defined haplotype encompassing CCL3 for sequence analysis.
2. Material and methods 2.1. Patients and families Altogether, 17 affected and 8 unaffected unrelated individuals were identified with the MS associated threemarker haplotype encompassing CCL3 in 1369 individuals from 361 trio, affected sibpair, incomplete and multiplex Caucasian families (details in Vyshkina and Kalman, 2005). All positive individuals were heterozygous for this haplotype defined by the A – T – C alleles of SNPs 278 –277 –273. 2.2. Automatic sequencing A 3500-kb DNA segment, including the MS-associated three-marker haplotype (1180 bp) and its 5V(1750 bp) and 3V (570 bp) flanking regions, was amplified by PCR in 7 overlapping fragments. An additional pair of primers was designed to avoid the interference of an insertion/deletion [between nt1487 and nt1495] with the annealing of the colocalizing forward primer in PCR segment 5 in some individuals. The DNA fragments were directly sequenced on an ABI Prism 3700 DNA Analyzer (Genaissance Pharmaceuticals, Inc., New Haven, CT). Sequences were aligned with the corresponding segment of the NC_010799 contig on chromosome 17 (http://www.ncbi.nlm.nih.gov/ entrez/viewer.fcgi?val=37544509&view=gbwithparts). 2.3. Genotyping SNPs revealed by sequencing were genotyped using the Sequenom MassARRAY System in the second set of 361 families. Associations of these alleles and haplotypes with MS were tested by using the pedigree disequilibrium test
217
(PDT), Family Based Association test (FBAT), the TRANSMIT version 2.5 program and Haplotype Based Association Test (HBAT) as previously described (Vyshkina and Kalman, 2005).
3. Results and discussion In search for a MS-related mutation, we sequenced an extended MS-associated haplotype defined by SNPs 278– 277 – 273 encompassing the CCL3 gene. This haplotype was only found in heterozygous state in 17 affected and 8 unaffected individuals out of 1369 in 361 families (Vyshkina and Kalman, 2005). 5Vand 3Vto the core haplotype within the extended region subjected to sequencing, there were two additional SNPs (280 and 271) that showed no direct association with the disease in the previous family studies (Fig. 1) (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). Similarly, none of the densely placed markers 5V or 3V to the selected 3500 bp DNA segment within the chromosomal block of linkage disequilibrium (LD) revealed association with MS in either of the previous studies (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). Therefore, we postulated that if a MS-relevant mutation is present, it must be within or in the proximity of the haplotype defined by SNPs 278 – 277– 273. Nevertheless, sequencing of the selected region revealed no new mutation, but 16 SNPs and 2 insertion/deletion variants in the 25 affected and unaffected individuals with the haplotype. All but two individual variants within the MS-associated chromosomes can be found in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/). Six of 16 SNPs were included but did not show association with MS in the previous family studies (Vyshkina and Kalman, 2005), and one insertion/deletion appeared only in the non-MS-associated chromosomes. Thus, we excluded these 9 variants from further genotyping and association analyses. The remaining variants were genotyped in 1369 individuals, in 361 families (described in Vyshkina and Kalman, 2005). The allele distribution of these markers was in agreement with the Hardy-Weinberg equilibrium, and none of them showed association with the disease using PDT and FBAT. In the TRANSMIT and HBAT analyses of pair-wise two-marker
Fig. 1. The sequenced 3500 base pair region with all variations revealed. The horizontal bar represents the sequenced 3500 bp DNA segment including the MS associated haplotype and its flanking regions. Introns, exons and the 5Vand 3VUTRs of CCL3 are indicated. The 16 SNPs and one insertion/deletion variant detected within the MS associated chromosomes in 25 individuals are designated by dots within the DNA symbol. Black dots represent SNPs 280, 278, 277, 274, 273 and 271 involved in previous genotyping and association studies. The three larger dots represent SNPs 278, 277 and 273 defining the MS associated haplotype. The SNP between markers 278 and 277 had a minor allele frequency of 0.002, and thus was not informative in the analyses.
218
T. Vyshkina, B. Kalman / Journal of Neuroimmunology 176 (2006) 216 – 218
haplotypes, an allelic combination of markers encompassing the 3VUTR of CCL3 and its 3V flanking region showed association in the HBAT ( p = 0.012), but this association was weak and the haplotype fell within the 3V segment of haplotype 278 – 277 – 273 (for which, a TRANSMIT p = 0.0023 and a HBAT p = 0.0078 was previously detected in the same set of families) (Vyshkina and Kalman, 2005). After completing the association analyses of all variants and haplotypes in this region, the strongest and most consistently observed finding remained the association with the A – T allelic combination of SNPs 278– 277 (TRANSMIT p = 0.0001, HBAT p = 0.0045) in the exon II and intron I of CCL3 (Fig. 1). In summary, sequence analyses of an MS associated haplotype and its flanking regions encompassing the gene of CCL3 revealed no new mutation. The detected sequence variations also did not show association with the disease when genotyped and tested for transmission distortion in families. Although the existence of a disease relevant variant in LD with the investigated extended haplotype may be postulated, it is not likely given the lack of SNP or haplotype associations in two independent studies with dense marker pacing in the surrounding LD block (Vyshkina et al., 2005; Vyshkina and Kalman, 2005). After these sequential and comprehensive analyses, the strongest positive finding remains the association of haplotype 278A – 277T with MS in the CCL3 gene. Taken into consideration the established role of CCL3 in plaque development and the exclusion of other genetic markers, this haplotype itself may indicate a functionally important allelic interaction in MS.
Acknowledgments This study was supported by the RG 3521-A-6 grant from the National Multiple Sclerosis Society.
References Banisor, I., Leist, T.P., Kalman, B., 2005. Involvement of h-chemokines in the development of inflammatory demyelination. J. Neuroinflamm. 2, 7 (Electronic publication). Kantarci, O.H., Morales, Y., Ziemer, P.A., Hebrink, D.D., Mahad, D.J., Atkinson, E.J., Achenbach, S.J., De Andrade, M., Mack, M., Ransohoff, R.M., Lassmann, H., Bruck, W., Weinshenker, B.G., Lucchinetti, C.F., 2005. CCR5Delta32 polymorphism effects on CCR5 expression, patterns of immunopathology and disease course in multiple sclerosis. J. Neuroimmunol. 169, 137 – 143. Lucchinetti, C., Bruck, W., Parisi, J., Scheithauer, B., Rodriguez, M., Lassmann, H., 2000. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707 – 717. Mahad, D.J., Trebst, C., Kivisakk, P., Staugaitis, S.M., Tucky, B., Wei, T., Lucchinetti, C.F., Lassmann, H., Ransohoff, R.M., 2004. Expression of chemokine receptors CCR1 and CCR5 reflects differential activation of mononuclear phagocytes in pattern II and pattern III multiple sclerosis lesions. J. Neuropathol. Exp. Neurol. 63, 262 – 273. The Transatlantic Multiple Sclerosis Genetics Cooperative, 2001. A metaanalysis of genomic screens in multiple sclerosis. Mult. Scler. 7, 3 – 11. Vyshkina, T., Kalman, B., 2005. Haplotypes within genes of h-chemokines are associated with multiple sclerosis: a second phase study. Hum. Genet. 118, 67 – 75. Vyshkina, T., Shugart, Y.Y., Birnbaum, G., Leist, T.P., Kalman, B., 2005. Association of haplotypes in the beta-chemokine locus with multiple sclerosis. Eur. J. Hum. Genet. 13, 240 – 247.