We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT.

In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA.

No abstract

The species diversity of cichlids was investigated in Kwan Phayao from August 2016 to May 2017. Four cichlid species were found, including Oreochromis niloticus, Oreochromis mossambicus, Coptodon rendalli and Coptodon zillii. Due to similar characterizations, it is very difficult to identify each species. Three molecular methods were used to distinguish these four species. DNA barcodes or partial cytochrome c oxidase I (COI) gene sequences were amplified by PCR and sequenced. In Oreochromis sp. and Coptodon sp., 707- and 704-bp fragments were amplified, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis showed clear differences between the four cichlid species after digestion with three restriction enzymes, ScaI, HindIII and PdiI. ScaI and HindIII separated Oreochromis sp. from Coptodon sp. due to different fragment sizes. PdiI distinguished each cichlid species in the same genus. Finally, high resolution melting (HRM) analysis showed the sensitivity of the primers for discriminating these species with small amplicons and melting curves. From the comparison, HRM analysis was the most efficient method because the primer was shown to be sensitive for discriminating the four cichlids. In addition, it was inexpensive and required a short time to detect large samples. However, direct sequencing or DNA barcodes were still necessary in the case of the COI sequences of organisms of interest, which have not been reported in any databases. These four cichlids are alien species in Thailand; thus, species identification is very important for fishery management.