Prepit l2p

A commercial kit (prepIT-L2P, DNA Genotek, Ottawa, ON, Canada) was used to extract DNA from each saliva sample, collected with a specific Oragene DNA tube at the first time point of the field study, after which the DNA was stored at −20 °C. The DNA was then analyzed with commercial genotyping assays for the SNP CC16 G38A polymorphism (rs3741240) and SNP UGRP1 G112A (rs1368408) as previously described [32 (link)].

Children under 16 years of age attending blind schools in Bhutan (Farmer et al., 2015), Cambodia (Sia et al., 2010), and Sri Lanka (Gao et al., 2011) underwent an ocular examination and review of records as part of audits of the causes of childhood blindness in each community as described previously (Farmer et al., 2015; Gao et al., 2011; Sia et al., 2010). While all children were examined at the time of recruitment, it was not possible to access historical medical records to determine age of onset, or to interview the family for a detailed history. The recruitment of additional family members was only possible if the family decided to attend the school on the day of the survey.
Patients were included in this analysis if they were observed to have bilateral pediatric (or congenital) cataract, with or without other ocular or systemic features. Patients with aniridia were excluded, even if cataract was also present. Where possible, additional affected and unaffected family members were also examined and recruited. Saliva was collected from each participant in the DNA saliva collection kit (Oragene DNA saliva collection kit) and extracted using prepIT L2P (DNA Genotek Inc., Ottawa, ON, Canada).

Genomic DNA was extracted from blood using the Puregene Blood Core Kit (QIAGEN), saliva using the Oragene Saliva Kit (DNA Genotek), buccal cells from Oragene Saliva Kits following centrifugation, and skin fibroblasts following a skin biopsy. Fibroblasts were grown in DMEM/F12 with glutamine, supplemented with 10% FBS and 1% PenStrep (all three from Gibco). DNA was extracted both directly and from fibroblasts after digesting with proteinase K in Cell Lysis Buffer (both QIAGEN) using prepIT.L2P (DNA Genotek) and ethanol precipitation. Lymphocytes were separated from whole blood using Ficoll and subsequently transformed to lymphoblastoid (LB) cell lines using Epstein-Barr virus (EBV) in RPMI 1640 media with glutamine, supplemented with 10% FBS and 1% PenStrep (all three from Gibco). Genomic DNA was then extracted using a Puregene Blood Core Kit.

DNA was collected from saliva using Oragene-DNA kits (DNA Genotek Inc.) and extracted with prepIT-L2P (DNA Genotek Inc.) using manufacturer protocols. READ1 genotyping was conducted using PCR amplification and purified of PCR reagents. Sanger sequencing was performed at the Yale W.M. Keck DNA Sequencing Facility using standard protocols. Primer sequences and amplification protocols are described in detail elsewhere.^{30 (link)} READ1 genotypes were called from chromatograms using a custom program written in C + + (Dr. Yong Kong, available upon request). If the calling program identified errors, chromatograms were manually examined and deconvoluted for genotype calls. The genotype call rate for READ1 alleles in the GRaD sample was 0.987.
Allele-specific PCR was used to genotype the 2445 bp microdeletion in intron 2 of DCDC2, which encompasses READ1. Primer sequences and amplification protocols for microdeletion genotyping are described in detail elsewhere.^{30 (link)} The deletion genotype call rate was 0.972 in the GRaD sample.

Saliva samples were incubated for 2 h at 56 °C, and DNA isolation carried out with PrepIT.L2P (DNA Genotek Inc., Canada) as per the manufacturer’s instructions. The purity and integrity of the genomic DNA preparations were assessed by agarose gel electrophoresis, and the quantity of DNA was determined using Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Paisley, UK). In preparation for DNA methylation analysis, 500 ng of DNA was bisulphite converted using the EZ DNA Methylation Kit (Zymo Research, CA, USA) according to manufacturer’s instructions. Genome-wide DNA methylation profiles were generated using the Infinium Methylation EPIC Beadchip Array, and the Beadchip images captured using an Illumina iScan (Cambridge Genomic Services, Cambridge, UK) for matched cases (n = 16) and controls (n = 16).