FCGR2B rs1050501 - ASHG 2012 - 22October2012

Jennifer M. Reynolds

Clinical Pharmacogenomics Laboratory – Pharmacokinetics, Dynamics, and Metabolism Dept.

Pfizer, Inc., Groton, CT, USA.

Contact : [email protected]

BACKGROUND The FCGR2B rs1050501 polymorphism has been associated with SLE (Systemic Lupus Erythematosus) in several studies of Asian populations (1-4), but

inconsistently associated with SLE in Caucasians (5-7). After examining the rs1050501 genotyping methods used in these studies, it was observed that many of

of the papers (4-6) used a forward primer annealing to a location with two putative SNPs, rs2793082 and rs2793081 . To determine if the SNPs reported were

real variation or single nucleotide disagreements between FCGR2B and the highly homologous FCGR2C, the rs1050501 region of FCGR2B was resequenced

from an ethnically diverse panel. The putative SNPs were absent in Chinese and Yoruba samples, but appeared to be common in Caucasians.

To determine if the SNPs played a role in genotyping accuracy of rs1050501, a subset of the HapMap01 Panel (Caucasian) and HapMap02 Panel (Chinese and

Japanese) were genotyped for rs1050501 using two assays identical except for the forward primer. One assay used the published forward primer location

containing the rs2793082 and rs2793081 SNPs, while the second assay used a FCGR2B specific forward primer location identified from the resequencing

results of the ethnically diverse panel. Both panels were also genotyped for rs2793082 and rs2793081. The rs1050501 genotyping method employing a forward

primer annealing to the location containing rs2793082 and rs2793081 affected genotyping accuracy in 14% of the Caucasian samples studied.

SUBJECTS AND METHODS

HapMap01 (Caucasian), HapMap02 (Chinese and Japanese) and HapMap03

(Yoruba) gDNA samples were obtained from Coriell. Eight subjects from each

ethnic group were resequenced for a 2.5 kb region of FCGR2B containing

rs1050501. HapMap01 and HapMap02 were genotyped for rs1050501 using a

TaqMan® assay with primers and probes which were chosen from the non-

polymorphic regions of FCGR2B (Non-Polymorphic rs1050501 assay) as

identified by the resequencing efforts. The same set of samples was also

genotyped with a second rs1050501 TaqMan® genotyping assay identical to

the first, with the exception of a forward primer annealing to rs2793082 and

rs2793081 (Polymorphic rs1050501 assay) . Close proximity of the rs2793082

and rs2793081 polymorphisms inhibited probe design, and therefore these

SNPs were genotyped from the HapMap01 and HapMap02 panels using a

sequencing assay (primers not shown in Fig.1).

CONCLUSION Use of the rs2793082 and rs2793081 region for a

forward primer location when genotyping rs1050501 in

Caucasians should be avoided due to the high

frequency of rs2793082 and rs2793081 in Caucasians,

and the ability of these SNPs to affect genotyping

accuracy in 14% of samples in a Caucasian panel.

Polymorphisms in Primer Binding Site affect Genotyping

Accuracy of FCGR2B rs1050501 in Caucasians

Figure 1. Schematic of amplifying primers on FCGR2B

RESULTS

Table 1. Resequencing Results - rs2793082 and rs2793081 were confirmed to be true variation

within FCGR2B. Controls for validating accuracy of genotyping assays were identified.

HGVS Nomenclature rs# Location

Caucasian

MAF

(n=8)

Chinese

MAF

(n=8)

Yoruba

MAF

(n=8)

NM_004001.4: c.647-243C>T rs2793082 Intron 4 25.0% 0.0% 0.0%

NM_004001.4: c.647-238T>C rs2793081 Intron 4 25.0% 0.0% 0.0%

NM_004001.4: c.695T>C rs1050501 Exon 5 12.5% 37.5% 12.5%

Table 2. Genotyping Results for Caucasian samples with discrepancies between the Non-Polymorphic and

Polymorphic rs1050501 Assays – All samples contain at least one copy of the variant alleles for rs2793082 and

rs2793081.

Sample Name Ethnicity rs2793082 (C->T) rs2793081 (T->C)

rs1050501 (T->C)

by Non-

Polymorphic

Assay

rs1050501 (T->C)

by Polymorphic

Assay

NA10830 Caucasian T/T C/C T/T Undetermined

NA10831 Caucasian C/T T/C T/C C/C











In samples where the rs1050501 Non-Polymorphic

and Polymorphic assays disagreed, the samples were

sequenced using the resequencing primers and the

Non-Polymorphic assay was confirmed to be accurate.

It can be seen from Table 2 that in all cases where the

two assays disagreed, the sample contained at least

one copy of the variant alleles for rs2793082 and

rs2793081. In samples heterozygous for rs2793082

and rs2793081, the Polymorphic assay could only

amplify from one allele. In samples that were

homozygous for rs2793082 and rs2793081, the

Polymorphic assay could not amplify from either allele.

Table 3. Genotyping Results - rs2793082 and rs2793081 are

common in Caucasian, rare in Japanese, and appear to be absent

in Chinese.

Caucasian (n=84) Chinese (n=44) Japanese (n=44)

rs2793082 25.6% 0.0% 1.1%

rs2793081 25.6% 0.0% 1.1%

rs1050501

(Non-Polymorphic

Assay) 11.9% 29.5% 13.3%

REFERENCES 1. Kyogoku et al. Fcγ Receptor Gene Polymorphisms in Japanese Patients With Systemic Lupus Erythematosus. Arthritis Rheum 2002 : 46: 1242-1254. 2. Siriboonrit et al. Association of Fcγ receptor IIb and IIIb polymorphisms with

susceptibility to systemic lupus erythematosus in Thais. Tissue Antigens 2003: 61:374-383. 3. Chu et al. Association of Fcγ receptor IIb polymorphism with susceptibility to systemic lupus erythematosus in Chinese: a common susceptibility gene in Asian

Populations. Tissue Antigens 2004:63:21-27. 4. Chen et al. Association of Transmembrane Polymorphism of Fcγ receptor IIb (FCGR2B) With Systemic Lupus Erythematosus in Taiwanese Patients. Arthritis Rheum 2006: 54: 3908-3917. 5. Floto et al. Loss of Function of a lupus-associated FcγRIIB polymorphism through exclusion from lipid rafts. Nat Med 2005. 11: 1056-1058. 6. Willcocks et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to

systemic lupus erythematosus. PNAS Early Edition 2010. 7. Magnussen et al. Polymorphisms of the Fcγ receptor type IIB gene are not associated with systemic lupus erythematosus in the Swedish population. Arthritis Rheum 2004: 50: 1348-1350.

Presented at ASHG 2012

FCGR2B rs1050501 - ASHG 2012 - 22October2012

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