Detection of genetic defect within ABCA3 from newborns with respiratory distress syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>To detect possible relationship between genetic defect within the gene encoding member A3 of the ATP Binding Cassette family (ABCA3) and neonatal respiratory distress syndrome (NRDS), thus to understand the genetic mechanisms of NRDS in Han ethnic group.</p><p><b>METHOD</b>The clinical data of 11 cases with NRDS hospitalized in neonatal intensive care unit was investigated. Blood samples were collected from 11 cases with NRDS and 97 unassociated normal individuals. Polymerase chain reaction (PCR) and DNA direct sequencing were performed to screen all exons and their flanking introns of ABCA3 gene for mutation analysis in 11 cases with NRDS. If a new missense variation was identified, single strand conformation polymorphism analysis was performed in 97 healthy controls. Lung tissue sample from a case who died 12 hours after birth was examined with light microscopy and electron microscopy.</p><p><b>RESULT</b>Three missense genetic variants in exons, which include c. 2169 G > A (p.M723I), c. 1010 T > G (p.V337G), c. 4972 A > G (p.S1658G), one splice junction site variation (Exon 30 + 2 T/G), several unreported polymorphism sites [213 C > T(p.F71F), exon 21 + 34C/T] and reported polymorphism site (p.F353F) were identified on ABCA3 gene coding region in 11 case. The homozygous variation (c.2169G > A), which was in exon 17 and causes an M723I amino acid change, was found in the case who died 13 hours after birth, but not detected in 97 controls, indicating that this variation is indeed a mutation and not a polymorphism. In the case carrying c.2169G > A, ultrastructural examination of the alveolar type II cells with electron microscopy demonstrated abnormally small and dense lamellar body with eccentrically distributed electron dense substance.</p><p><b>CONCLUSION</b>Genetic variants within ABCA3 may be the genetic cause of or a contributor to some unexplained refractory NRDS. Identification of ABCA3 genetic variant in NRDS infants is important to establish appropriate management and evaluation of treatment options, as well as to offer genetic counseling and prenatal diagnosis.</p>

Site-directed mutagenesis and protein expression of KCNQ2 gene associated with neonatal convulsions / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the protocol of construction of a KCNQ2-c.812G>T mutant and it's eukaryotic expression vector, the c.812G>T (p.G271V) mutation which was detected in a Chinese pedigree of benign familial infantile convulsions, and to examine the expression of mutant protein in human embyonic kidney (HEK) 293 cells.</p><p><b>METHODS</b>A KCNQ2 mutation c.812G>T was engineered on KCNQ2 cDNAs cloned into pcDNA3.0 by sequence overlap extension PCR and restriction enzymes. HEK293 cells were co-transfected with pRK5-GFP and KCNQ2 plasmid (the wild type or mutant) using lipofectamine and then subjected to confocal microscopy. The transfected cells were immunostained to visualize the intracellular expression of the mutant molecules.</p><p><b>RESULTS</b>Direct sequence analysis revealed a G to T transition at position 812. The c.812G>T mutation was correctly combined to eukaryotic expressive vector pcDNA3.0 and expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both the wild type and mutant molecules on the plasma membrane, which suggested that the c.812G>T mutation at the pore forming region of KCNQ2 channel did not impair normal protein expression in HEK293 cells.</p><p><b>CONCLUSIONS</b>Successful construction of mutant KCNQ2 eukaryotic expression vector and expression of KCNQ2 protein in HEK293 cells provide a basis for further study on the functional effects of convulsion-causing KCNQ2 mutations and for understanding the molecular pathogenesis of epilepsy.</p>