METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.
CASE PRESENTATION: This is a rare case of a pair of 8-year-old monochorionic diamniotic identical twin, who on family cascade screening were diagnosed as definite FH, according to the Dutch Lipid Clinic Criteria (DLCC) with a score of 10. There were no lipid stigmata noted. Baseline lipid profiles revealed severe hypercholesterolaemia, (TC = 10.5 mmol/L, 10.6 mmol/L; LDL-c = 8.8 mmol/L, 8.6 mmol/L respectively). Their father is the index case who initially presented with premature CAD, and subsequently diagnosed as FH. Family cascade screening identified clinical FH in other family members including their paternal grandfather who also had premature CAD, and another elder brother, aged 10 years. Genetic analysis by targeted next-generation sequencing using MiSeq platform (Illumina) was performed to detect mutations in LDLR, APOB100, PCSK9, ABCG5, ABCG8, APOE and LDLRAP1 genes. Results revealed that the twin, their elder brother, father and grandfather are heterozygous for a missense mutation (c.530C > T) in LDLR that was previously reported as a pathogenic mutation. In addition, the twin has heterozygous ABCG8 gene mutation (c.55G > C). Their eldest brother aged 12 years and their mother both had normal lipid profiles with absence of LDLR gene mutation.
CONCLUSION: A rare case of Asian monochorionic diamniotic identical twin, with clinically diagnosed and molecularly confirmed heterozygous FH, due to LDLR and ABCG8 gene mutations have been reported. Childhood FH may not present with the classical physical manifestations including the pathognomonic lipid stigmata as in adults. Therefore, childhood FH can be diagnosed early using a combination of clinical criteria and molecular analyses.
MATERIALS AND METHODS: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification.
RESULTS: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR.
CONCLUSION: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.