Résumé
Background & objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of ?-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for ?-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma. Methods: Pregnant women and their spouses who are at risk of bearing an offspring with ?-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited ?-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with ?-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. Interpretation & conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal ?-thalassaemia in at risk pregnancies.
Résumé
The outbreak of highly pathogenic avian influenza (HPAI) viruses has severely disrupted poultry production and trade. Humans have been infected with HPAI H5N1 viruses and many have died. The nonstructural (NS) proteins of the virus are a factor that determines virulence. In this report, 80 NS genes of H5N1 HPAI viruses isolated from Thailand were completely sequenced and phylogenically analyzed. The percentages of identity and variable site NS1 genes were similar to NS2/nuclear export protein (NEP) genes. All NS1 genes from the samples were located in allelic group A. The NS1 and NS2/NEP proteins possess 225 and 121 amino acids, respectively. All NS1 protein samples had five amino acid deletions typical of avian influenza viruses isolated since 2002. An amino acid substitution at position 92 (G92E) of the NS1 protein, known to promote the inhibition of host immune responses, was also found in the samples.