Buccal Swab Analysis of Mitochondrial Enzyme Deficiency and DNA Defects in a Child With Suspected Myoclonic Epilepsy and Ragged Red Fibers (MERRF)
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The authors describe mitochondrial studies in a 6-year-old patient with a seizure disorder that can be seen in myoclonic epilepsy and ragged red fibers. Using a recently developed noninvasive approach, analysis of buccal mitochondrial enzyme function revealed severe respiratory complex I and IV deficiencies in the patient. In addition, analysis of buccal mitochondrial DNA showed significant amounts of the common 5 kb and 7.4 kb mitochondrial DNA deletions, also detectable in blood. This study suggests that a buccal swab approach can be used to informatively examine mitochondrial dysfunction in children with seizures and may be applicable to screening mitochondrial disease with other clinical presentations.Keywords:
Buccal swab
Myoclonic epilepsy
Mitochondrial disease
Abstract Background: The human microbiota is postulated to affect cancer risk, but collecting microbiota specimens with prospective follow-up for diseases will take time. Buccal cell samples have been obtained from mouthwash for the study of human genomic DNA in many cohort studies. Here, we evaluate the feasibility of using buccal cell samples to examine associations of human microbiota and disease risk. Methods: We obtained buccal cells from mouthwash in 41 healthy participants using a protocol that is widely employed to obtain buccal cells for the study of human DNA. We compared oral microbiota from buccal cells with that from eight other oral sample types collected by following the protocols of the Human Microbiome Project. Microbiota profiles were determined by sequencing 16S rRNA gene V3–V4 region. Results: Compared with each of the eight other oral samples, the buccal cell samples had significantly more observed species (P < 0.002) and higher alpha diversity (Shannon index, P < 0.02). The microbial communities were more similar (smaller beta diversity) among buccal cells samples than in the other samples (P < 0.001 for 12 of 16 weighted and unweighted UniFrac distance comparisons). Buccal cell microbial profiles closely resembled saliva but were distinct from dental plaque and tongue dorsum. Conclusions: Stored buccal cell samples in prospective cohort studies are a promising resource to study associations of oral microbiota with disease. Impact: The feasibility of using existing buccal cell collections in large prospective cohorts allows investigations of the role of oral microbiota in chronic disease etiology in large population studies possible today. Cancer Epidemiol Biomarkers Prev; 26(2); 249–53. ©2016 AACR.
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Abstract Background Telomere length (TL) in blood has been extensively studied as a biomarker of aging and aging-associated disease. TL in blood cells is commonly used as a proxy for TL in other tissue types. The source of DNA of adequate quality and quantity is an important consideration in telomere length analysis. Compared to blood cells, buccal cells easy for genomic DNA preparation would facilitate the rapid and reliable telomere length analysis. However, the feasibility of buccal cells for TL analysis remains yet unestablished. Methods A total of 52 participants ranged in age from 18 to 80 years including 24 males and 28 females were included in this study. Both buccal and blood samples were taken at the same time by using buccal cell swabs and fingertip stick from each participant. Relative telomere length (RTL) was analyzed using the quantitative real-time polymerase chain reaction (qPCR) method. Results The results indicate that there is a strong positive correlation between buccal RTL and blood RTL and negative correlation between both buccal RTL and blood RTL with age. Conclusion The validity of sampling using buccal cell swabs provides simple operation and good reproducibility for telomere length analysis, which overcomes the discomfort and risk of infection caused by blood sampling.
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Background – There is a need to develop minimally invasive methodologies to measure changes in tissue nutrient levels and alterations in disease risk biomarkers. Objective – We aimed to evaluate the use of buccal cells as a non-invasive approach to measure nutrient levels (selenium, zinc and magnesium) and markers of oxidative stress and accelerated ageing (telomere length) in crosssectional study. Design – Buccal cells and blood samples were collected from 91 volunteers. This cohort comprised 18M and 25F in the young group (aged 18-31 years), and 25M and 23F in the older group (65-75yrs). Se, Zn, Mg and Ca were measured in serum and buccal cells (by ICPMS and ICPOES). Telomere length measures were determined in lymphocytes and buccal cells (by Flow and qRTm-PCR). Outcomes – Only Se levels were significantly correlated in serum and buccal cell samples (r=0.3, p<0.01). Serum Ca was negatively correlated with buccal cell Ca (r=-0.25, p<0.01). There were significant correlations of age with: serum Se (r=0.27, p<0.01) and buccal Ca (r=0.24, p<0.05), buccal Se (r=0.28, p<0.01). Telomere length correlated negatively with age in lymphocytes (r=-0.28, p<0.01) and positively with age in the buccal cells (r=0.22, p<0.05). Conclusions -–Trace elements and telomere length can be measured in buccal samples. However, with the exception of Se, results in cells were not positively correlated with each other. These data indicate that the measurements in plasma and buccal samples are not interchangeable. The positive correlation of telomere length with age in buccal cells indicates a strong difference from lymphocytes and suggests differences in telomere length dynamics in the haematopoietic and epithelial tissue.
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Neonatal buccal cell collection for DNA analysisIt is considered undesirable to take blood from an infant as a means of obtaining DNA for research purposes.Sufficient DNA for direct gene analysis can be obtained from adult buccal epithelial cells, 1 but there is no evidence that neonatal buccal epithelial cells can similarly be used.We have carried out a preliminary study, approved by the Riverside Research Ethics Committee, to determine the ease of isolating neonatal buccal cells for DNA extraction followed by polymerase chain reaction (PCR) and sequence analysis.Buccal cells were obtained from eight term and four preterm infants after informed parental consent.A standard microbiological cotton swab or cotton dental roll was rubbed on the inner cheek, and the baby was allowed to suck on it for 30 seconds.The infants were unperturbed by the procedure and mothers found it very acceptable.Swabs/dental rolls were placed in 0.9% sodium chloride and centrifuged to concentrate the cells into a pellet.The cells were lysed with a standard lysis buffer, and DNA was extracted using a simple phenol/chloroform technique. 2Spectrophotometry to mea- sure the A 260 /A 280 ratio in order to assess purity of the extracted DNA was carried out using an Eppendorf Biophotometer V1.20.DNA concentration and yield were calculated from UV absorbance at 260 nm (A 260 ).To show that the DNA obtained could be analysed at the single base level, PCR was carried out with the primers AACACTGGTGG CGCAGAAAT (forward) and TGGGTGCACCT CTCACAGAA (reverse) to amplify exon 22 of the human SCRIBBLE gene.PCR products were viewed on a 1% agarose gel with a UV transilluminator, sequenced using BigDye v3.0 chemistry (PE Applied Biosystems, Warrington, UK) and run on an ABI 3100 Genetic Analyser.Sequence alignment with the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to confirm accurate sequence amplification.Spectrophotometry confirmed successful DNA extraction from all samples with a mean DNA yield of 6.65 mg (SD 4.07; range 1. 65-16.40).This compares favourably with DNA yields obtained in similar studies with adult and child subjects where yields of 2-85 mg have been described. 3 4Sequences matching exon 22 of SCRIBBLE were successfully obtained using both forward and reverse primers.The mean A 260 /A 280 ratio of samples in this work was 1.27 (SD 0.07; range 1.20-1.42).High quality DNA has an A 260 /A 280 ratio of about 1.80.This preliminary study has shown that buccal cells can easily be obtained from term and preterm infants in sufficient quantity for DNA extraction, PCR, and sequencing.The technique is simple and non-invasive and we hope will facilitate neonatal research.
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Background: Infections can be/are a global factor of mutational damage, causing undesirable changes in heredity both in the present and in future generations. All this makes the problem of infectious mutagenesis extremely urgent and requires timely assessment of the mutagenic effect and therapeutic and preventive assistance to the population in highly pathogenic viral infections. Aim: the study evaluated the effect of COVID-19 on the genetic apparatus of buccal epithelial cells in people who had undergone coronavirus infection of varying severity. Methods: The genetic status of people 2-3 months after infection with COVID-19 was studied by karyological and micronucleus analysis in buccal epithelial cells of the oral cavity. Results and Discussion: Reliable increase of karyological indices (cytogenetic disorders (micronuclei), proliferation indices, nucleus destruction indices) in the buccal epithelium of the oral cavity was detected. The studied abnormalities were detected in all patients who underwent the disease regardless of disease severity and contacts. In patients with the severe form of COVID-19, a significant increase in cytogenetic abnormalities was noted in comparison with the data of the control group and the indices of patients with moderate and mild forms of the disease. Conclusions: contact with the SARS-CoV-2 virus, regardless of the presence of clinical manifestations, causes karyological abnormalities in the buccal epithelium of the oral cavity. This indicates its significant mutagenic potential.
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Abstract Collection and analysis of DNA, most commonly from blood or buccal cells, is becoming more common in epidemiologic studies. Buccal samples, which are painless to take and relatively easily collected, are often the preferred source. There are several buccal cell collection methods: swabs, brushes, mouthwash, and treated cards, such as FTA or IsoCode cards. Few studies have systematically compared methods of buccal cell collection with respect to DNA yield and amplification success under similar conditions. We compared buccal DNA collection and amplification using buccal swabs and FTA cards in 122 control subjects from our Australian case-control study of childhood acute lymphoblastic leukaemia. Buccal DNA was quantified using a real-time PCR for β-actin and genotyped at the loci of three polymorphisms (MTHFR 677C>T, ACE I/D, and XPD 1012G>A). PCR was successful with DNA from buccal swabs for 62% to 89% of subjects and from FTA cards for 83% to 100% of subjects, depending on the locus. The matched pair odds ratios (95% confidence interval) comparing success of FTA cards with buccal swabs are as follows: MTHFR 677C>T using PCR-RFLP, 12.5 (11.6-13.5) and using real-time PCR, 130.0 (113.1-152.8); ACE I/D using PCR-amplified fragment length polymorphism, 3.36 (3.2-3.5); XPD 1012G>A using real-time PCR, 150.0 (132.7-172.3). FTA cards are a robust DNA collection method and generally produce DNA suitable for PCR more reliably than buccal swabs. There are, however, technical challenges in handling discs punched from FTA cards that intending users should be aware of. (Cancer Epidemiol Biomarkers Prev 2006;15(4):816–9)
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