Background Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility.
Methods We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues.
Results A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma.
Conclusion SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.
- complex traits
- genetic epidemiology
- infectious diseases
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Collaborators University of Washington Center for Mendelian Genomics.
Contributors RLPS-C conceptualised the study. TKLY, MP, PJL, MLCT, MRTR-Q, EGdVL, RARdlC, RMAN, KMCO, JPMM, ANEC, MCE-C, MLSA, TLGC, GTA, EMC-dlP, ALC, CMC and RLPS-C collected clinical data, saliva samples and microbial swabs from the indigenous Filipino population and PGH patients. WS, HP and TC collected clinical data and saliva samples from Texan families. LH, EE, JK and PSM provided clinical data and DNA samples from Finnish families. KAD and MMS provided clinical data and DNA samples from Minnesota families. NRL and KLM provided DNA samples from the Cebu Longitudinal Health and Nutrition Survey cohort. TCB, MJS and NA performed human and microbial DNA isolation, PCR and Sanger sequencing. APJG, SY, SR and ZMA performed immunolocalisation experiments and sequencing for Pakistani families. AFR provided microarray expression data from mouse ME. The University of Washington Center for Mendelian Genomics, MJB, DAN and SML performed exome sequencing of DNA samples from indigenous Filipinos and 12 Minnesota families. DNF, DI and CER performed 16S rRNA sequencing and analyses on microbial samples. RLPS-C performed exome, linkage and statistical analyses for variant data. DNF and RLPS-C wrote the manuscript. All authors read, provided critical input and approved the final version of the manuscript.
Funding The Cebu Longitudinal Health and Nutrition Survey was supported by NIH grants DK078150, TW005596, HL085144 and TW008288 and pilot funds from RR020649, ES010126 and DK056350. Exome sequencing of DNA samples from the indigenous Filipino population and 12 Minnesota families was provided by the UW-CMG and was supported by the US NIH-National Human Genome Research Institute and the National Heart, Lung and Blood Institute grants UM1 HG006493 and U24 HG008956 (to DAN, MJB and SML). This study was supported by the Philippine Council for Health Research and Development - Department of Science and Technology Balik Scientist Program and the US NIH – National Institute on Deafness and Other Communication Disorders grant R01 DC015004 (both to RLPSC).
Disclaimer Members of the University of Washington Center for Mendelian Genomics are listed in https://uwcmg.org/docs/Crediting_UW-CMG/UW_CMG_Banner.pdf.
Competing interests AR is a cofounder of, shareholder in and uncompensated consultant to Otonomy, Inc, a relationship that was approved by the University of California San Diego.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available in a public, open access repository. Novel variants are cited in ClinVar with accession numbers SCV001338935-SCV001338943. Demultiplexed paired-end 16S rRNA sequence data were deposited in the NCBI Short Read Archive under accession number PRJNA439435. RNA-sequence data from cholesteatoma and mucosal samples from OM patients  are available in dbGaP:phs001941.v1.p1.
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