The current method of obtaining DNA for genetic evaluation in Very Low Birthweight infants requires obtaining a blood sample. In these premature neonates, every milliliter of blood drawn is significant in terms of their total blood volume. It is the purpose of this study to evaluate if a non-invasive test can be used as a substitute to obtain enough high-quality DNA to perform genetic analysis including single nucleotide polymorphism (SNP) genotyping, evaluation of copy number variants, and exome sequencing.
We hypothesize that genetic analysis of premature infants with a non-invasive test, specifically a buccal swab, results in sufficient, high quality DNA to determine their genotype for a SNP associated with a hyper-immune state.
Patients were prospectively recruited if they were admitted to the NICU at less than 34 weeks gestation, or transferred in with a diagnosis of necrotizing enterocolitis (NEC). Patients were excluded if they had congenital heart disease (except a Patent Ductus Arteriosus), or a confirmed diagnosis of congenital anomaly, genetic disorder, or inborn error of metabolism. We collected both blood and 2 buccal swabs from each enrolled patient. DNA was extracted from the buccal swab using the Qiagen Gentra Puregene Buccal Cell Kit. The minimum required amount of DNA needed to perform genotyping for a restriction fragment length polymorphism (RFLP) is 5 ng in our lab. We amplified 200 ng of template DNA using the polymerase chain reaction (PCR) and used gel-based RFLP to determine a genotype.
A total of 44 buccal swabs were obtained from 22 patients. DNA was extracted and yield measured using NanoDrop spectroscopy at wavelengths of 260 and 280 nm revealing an average 260/280 ratio of 1.80 (± 0.12). An average DNA concentration of 347 ng/µl (range 32-2424 ng/µl) was obtained. The DNA was used in a PCR-RFLP assay to successfully genotype a SNP. We confirmed the genotype results using standards with sequencing-determined genotypes. In addition, we duplicated the PCR-RFLP and obtained the same genotypes for 100% concordance.
Genomic DNA extraction from cheek cells obtained by buccal swabs in premature neonates is feasible, simple, and non-invasive. This method can potentially be used as an alternative to drawing blood for genetic analysis. Further studies will aim to validate this method against the current gold standard of a blood specimen.