Oragene saliva kit

Individuals with a confirmed diagnosis of otosclerosis were recruited from the Royal National Throat Nose and Ear Hospital, London, UK and The Princess Margaret Hospital in Windsor. The study was approved by the London Bloomsbury NRES Ethics committee (11/LO/0489) and patients were recruited by informed consent. From this cohort, a sub-cohort of individuals with evidence of familial otosclerosis was identified based on patient questionnaire data of family history (defined as two or more relatives with otosclerosis). Blood or saliva samples (Oragene® Saliva Kit, DNA Genotek) were obtained for genomic DNA isolation by standard methods. For those patients undergoing stapedotomy surgery to remove the stapes superstructure, the tissue was retained for gene expression studies. Control stapes were obtained from individuals where the stapes was removed as part of surgery for a number of other indicators including head trauma, glomus tumour and during total petrosectomy.

Saliva samples were collected using the Oragene Saliva kit, and DNA was extracted using the prepIT.L2P kit, both from DNA Genotek (Ottawa, Canada). DNA integrity was checked by agarose gel electrophoresis and quantitated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Further quantification was done using Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) as needed, following the manufacturer’s instructions.

Individuals with a confirmed clinical diagnosis of otosclerosis were recruited from the Royal National Throat Nose and Ear Hospital (London, UK), Princess Margaret Hospital (Windsor), Sunderland Royal Hospital (Sunderland), Freeman Hospital (Newcastle upon Tyne) and Ninewells Hospital (Dundee). The study was approved by the London Bloomsbury NRES Ethics committee (11/LO/0489). The diagnosis of otosclerosis was made on clinical and audiometric examination, and then confirmed during surgery. Patients and controls were recruited by informed consent. Controls had otosclerosis ruled out on the basis of a pure tone audiogram. Blood or saliva samples were obtained for genomic DNA isolation (Oragene^® Saliva Kit, DNA Genotek) by standard methods. Patients completed a questionnaire recording family history of otosclerosis and relevant medical history. Female patients answered additional questions recording history of pregnancy, oral contraceptives and hormone replacement therapy. For stratified analysis based on questionnaire data, patients were categorised as familial or non-familial based on reporting an additional first or second-degree family member with a clinical diagnosis of otosclerosis as reported by the proband.

Saliva samples were collected using the Oragene Saliva kit, and DNA was extracted using the prepIT.L2P kit, both from DNA Genotek (Ottawa, Canada). DNA integrity was checked by agarose gel electrophoresis and quantitated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Further quantification was done using Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) as needed, following the manufacturer’s instructions.

Genomic DNA was extracted from blood (Beijing ComWin Biotech), buccal mucosa (Beijing Zoman Biotechnology), saliva (Oragene Saliva Kit, DNA Genotek) or cultured cells (Qiagen DNeasy kit). Primers for polymerase chain reaction (PCR) amplification were designed using primer 3 (http://bioinfo.ut.ee/primer3) (Supplemental Table 1). After PCR amplification, 22 exons and flanking intron-exon boundaries of the ANO5 gene were Sanger sequenced using ABI PRISM 3730xl DNA Analyzers with standard protocol. Potential detrimental effects of missense variants were evaluated by in silico prediction tools PolyPhen2 and SIFT. Sequence alignment among different species was performed with ClustalW2.

The Hessequa descendant samples were collected in Western Cape, South Africa, after in-depth field work by an anthropologist (MDJ) on the ethnographic background of the communities in the region. Written informed consent was obtained from all 162 participants included in the study before saliva samples were collected. Sample collection of Coloured, Khoe-San and Khoe-San descendent groups were approved by the University of the Witwatersrand Human Research Ethics board, clearance numbers M980553, with renewals M050902, M090576, M1604104. This specific project was approved by University of the Witwatersrand Human Research Ethics board, clearance number M180655 and the National Ethics review board of Sweden, clearance number Dnr 2021–01448. The biological material was collected in 2 mL Oragene saliva kits (DNA Genotek) and DNA was extracted using the prepIT L2P extraction protocol. The 162 samples were genotyped on H3Africa Consortium SNP panel implemented in Illumina Infinium assay (H3Africa_2017_20021485_A2 BeadChip). The data were generated by the SNP&SEQ Technology Platform in Uppsala, Sweden. The data were analyzed using GenomeStudio v.2011.1 and aligned to the Human Genome built version 37. A total of 2,267,346 genomic markers were obtained.