ORIGINAL ARTICLE Association of the FBXO11 Gene With Chronic Otitis Media With Effusion and Recurrent Otitis Media The Minnesota COME/ROM Family Study Fernando Segade, PhD; Kathleen A. Daly, PhD; Dax Allred, BA; Pamela J. Hicks, BA; Miranda Cox, MS; Mark Brown, MS; Rachel E. Hardisty-Hughes, PhD; Steve D. M. Brown, PhD; Stephen S. Rich, PhD; Donald W. Bowden, PhD Objective: The FBXO11 gene is the human homo- analysis. In univariate genetic analysis, 1 reference SNP logue of the gene mutated in the novel deaf mouse mu- (hereinafter rs) (rs2134056) showed nominal evidence tant jeff (Jf), a single gene model of otitis media. We have of association to COME/ROM (P=.02), and 2 SNPs ap- evaluated single nucleotide polymorphisms (SNPs) in the proached significance (rs2020911, P=.06; rs3136367, FBXO11 gene for association with chronic otitis media P=.09). In multivariable analyses, including known risk with effusion/recurrent otitis media (COME/ROM). factors for COME/ROM (sex, exposure to smoking, at- tending day care centers, no prior breastfeeding, and hav- Design: A total of 13 SNPs were genotyped across the ing allergies), the evidence of independent association 98.7 kilobases of genomic DNA encompassing FBXO11. was reduced for each SNP (eg, rs2134056, from P=.02 Data were analyzed for single SNP association using gen- to P=.08). In subsequent analyses using the Pedigree Dis- eralized estimating equations, and haplotypes were evalu- equilibrium Test, the association of FBXO11 SNP ated using Pedigree Disequilibrium Test methods. rs2134056 (P=.06) with COME/ROM was confirmed. In- corporating multiple SNPs in 2- and 3-locus SNP hap- Patients: The Minnesota COME/ROM Family Study, a lotypes, those haplotypes containing rs2134056 also ex- group of 142 families (619 subjects) with multiple af- hibited evidence of association of FBXO11 and COME/ fected individuals with COME/ROM. ROM (P values ranging from .03 to .10). Main Outcome Measures: Genetic association of Conclusion: We have observed evidence consistent with COME/ROM with polymorphisms in FBXO11. an association between polymorphisms in FBXO11, the human homologue of the Jeff mouse model gene, and Results: The FBXO11 SNPs are contained in a single link- COME/ROM. age disequilibrium haplotype block. Ten of the 13 SNPs were sufficiently polymorphic in the sample to permit Arch Otolaryngol Head Neck Surg. 2006;132:729-733 HRONIC OTITIS MEDIA WITH ducted a linkage study (the Minnesota Author Affiliations: effusion (COME) and re- COME/ROM Family Study13) that re- Departments of Internal current otitis media vealed evidence for linkage to COME and Medicine (Drs Segade and (ROM) are relatively com- ROM on chromosome 10q and suggestive Bowden), Public Heath Sciences mon conditions, affect- evidence for linkage on chromosome 19q. (Ms Cox, Mr Brown, and ing 10% to 30% of children, and the rates Several case-control studies have identi- Dr Rich), and Biochemistry C 1-4 (Mr Allred, Ms Hicks, and of occurrence seem to be increasing. fied relationships between candidate genes Dr Bowden), and Center for These conditions often result in hearing and COME/ROM, including HLA anti- 5 6,7 14,15 16,17 Human Genomics (Drs Segade loss and middle ear sequelae. Disease gen, cytokine genes, and genes in- and Bowden, Mr Allred, and rates are highest in the preschool years and volved in the ability to clear infectious Ms Hicks), Wake Forest decline as children get older.1 These con- agents implicated in OM.18,19 To date, how- University School of Medicine, ditions are often treated with tympanos- ever, few detailed molecular genetic analy- Winston-Salem, NC; tomy tubes to ventilate the middle ear; this ses of genetic associations between candi- Department of Otolaryngology is the most common surgical procedure per- date genes and COME/ROM have been and Otitis Media Research formed in children in an ambulatory set- reported. Center, University of Minnesota ting.8 Epidemiologic studies2,9,10 have shown The FBXO11 gene is an intriguing can- School of Medicine, Minneapolis, (Dr Daly); and that a family history of these conditions in- didate for association with COME/ROM. Medical Research Council, crease a child’s personal risk for COME/ The deaf mouse mutant jeff represents a 11,12 20 Mammalian Genetics Unit ROM, and twin studies have demon- single gene model of COME, character- (Drs Hardisty-Hughes and strated a high degree of heritability for ROM ized by the development of OM in patho- Brown), Harwell, England. and otitis media (OM) duration. We con- gen-free conditions in the absence of any (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 132, JULY 2006 WWW.ARCHOTO.COM 729 ©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 other inflammatory disease. Animals with the jeff gene this study. We chose SNPs to cover the entire FBXO11 ge- (hereinafter, Jeff animals) present with fluid and pus ac- nomic sequence, including the 5Ј promoter region, exons, in- cumulation in the middle ear cavity, with diffuse muco- trons, and the 3Ј-untranslated region, and included rs2020911, sal inflammation, disruption of the eustachian epithe- rs3136367, rs3136371, rs3771285, rs3732191, rs330787, lium, and reduction of the lumen owing to abnormal rs2651767, rs2134056, rs960106, rs2937345, rs874869, 20 rs4952896, and rs7582252 (where rs indicates 1 reference SNP). growths. The jeff mutation appears to be fully pen- Of these SNPs, rs7582252, rs2937345, and rs3136371 were not etrant and has been mapped to the distal region of mouse polymorphic and were not included in subsequent analyses. 20 chromosome 17. A missense mutation has been iden- We performed genotyping of the FBXO11 SNP polymor- tified in Fbxo11, a gene located on this region of mouse phisms by employing the MassARRAY SNP genotyping sys- chromosome 17 (R. E. Hardisty-Hughes and S. D. M. tem (Sequenom Inc, San Diego, Calif) using a standard pro- Brown, unpublished data, 2006). The human ortholog, tocol.23 Total genomic DNA was purified from whole blood FBXO11, is located on chromosome 2 and encodes a 141- samples obtained from the subjects using PUREGENE DNA iso- residue protein that belongs to the F-box family of genes. lation kit (Gentra Inc, Minneapolis, Minn). We quantitated DNA FBXO11 is characterized by the presence of a 50–amino using standardized fluorometric readings on a Hoefer DyNA acid sequence, the F-box, which functions in protein- Quant 200 fluorometer (Hoefer Pharmacia Biotech Inc, San Fran- 21 cisco, Calif). Each sample was diluted to a final concentration protein interactions. The FBXO11 gene therefore rep- of 5 ng/µL. resents a strong candidate gene for OM. This article ex- amines polymorphisms in the FBXO11 gene for association in the human disease pathway for COME/ROM. STATISTICAL GENETIC ANALYSES METHODS We examined each pedigree for evidence of an incorrect fam- ily relationship by using genome scan data and PREST soft- ware.24 For each of the SNPs genotyped in this study, mende- PARTICIPANTS lian inconsistencies in their genotype assignment were examined using PEDCHECK software.25 Any genotypes inconsistent with All families and individuals in this study were recruited to par- 13 mendelian inheritance (9 total [0.001%]) were converted to miss- ticipate in the genetic linkage study. Families who were excluded ing. Following pedigree and genotype correction, maximum- from the linkage study because they lacked an affected sibling pair likelihood estimates of allele frequencies were computed us- with sufficient DNA were evaluated in this study. Study partici- ing the largest set of unrelated individuals. All SNPs were then pants included families from previous studies of OM conducted tested for departures from Hardy-Weinberg proportions. With by the Otitis Media Research Center at the University of Minne- multiple SNPs genotyped in the FBXO11 gene, the haplotype sota,Minneapolis,themembersofthegeneralpublicwhoresponded block structure and linkage disequilibrium (LD) between mark- to fliers posted around the university’s Academic Health Center, ers was assessed. Estimates of LD (DЈ and r2) were computed and otolaryngology clinic patients. Prospective families from pre- also using the largest set of unrelated individuals and Dprime vious studies were identified using OM history data collected in software. For families in which no founder was genotyped, the the original study on the participant (proband), parents, and sib- first affected offspring was used to estimate the statistics. lings. Mothers from the families recruited from the general pub- Association between individual SNP and COME/ROM sta- lic and clinic patients were interviewed about the OM history of tus was performed using a series of generalized estimating equa- all family members to determine family eligibility. Data were col- tion models.26 We adjusted for the correlation between sub- lected at the study visit to determine phenotype (history of chronic jects within a pedigree in the analyses by assuming exchangeable OM or ROM), including findings from the otomicroscopic exami- correlation among siblings within a pedigree and computing nation, multifrequency tympanometry, reported OM history, and 13 the sandwich estimator of the variance. The sandwich estima- medical record. An individual was considered to be affected if tor is also denoted the robust or empirical estimator of the vari- at least 2 of these sources had abnormal findings, or if 1 source ance and is robust to misspecification of the correlation ma- showed abnormalities and middle ear or tympanometric findings trix because it estimates the within-pedigree correlation matrix were presumed to be definitive evidence of a history of COME/ from the first and second moments of the data. Broadly speak- ROM (eg, tympanosclerosis, atrophy, or tympanogram with high ing, this method is comparable to a logistic regression analysis static admittance) were present. Subjects were examined by a neu- with adjustment for familial correlation among family mem- rotologist, and those with obvious craniofacial anomalies were ex- bers.