Résumé
Objective To investigate the role of TNSALP gene detection in prenatal diagnosis of HPP. Method The clinical data and the results of complete exon sequencing of TNSALP gene in one neonate with low alkaline phosphatase (HPP) were analyzed retrospectively. Peripheral bloods from his family members were collected. The amniotic fluid cell in fetuses at 17 weeks was tested for candidate gene mutations by Sanger sequencing. Results Mainly manifestations in 6-day-old baby were multiple fractures, limb shortening and bending and dyspnea. He died of respiratory failure 9 days after birth. The serum alkaline phosphatase was decreased and serum calcium was decreased slightly; serum phosphorus, serum 25 hydroxyvitamin-D and parathyroid hormone were normal. X-ray showed that the whole body bone was very poorly mineralized, and the long diaphysis was enlarged with shape of a cup at the end and multiple fractures existed. Gene sequencing revealed a complex heterozygous missense mutation in the TNSALP gene, including the heterozygous missense mutation c.542C>T in exon sixth causing 181st amino acids changed from serine to leucine (p.S181L), and tenth exon heterozygous missense mutation in c.1016G>A causing 339th amino acid changed from glycine to glutamic acid (p.G339E). The parental phenotypes were normal. The c.542C>T mutation is inherited from his father and the c.1016G>A mutation is inherited from his mother. These two mutations were not detected in the fetus. Conclusion TNSALP gene analysis can be applied to the diagnosis and prenatal diagnosis of HPP.
Résumé
Objective To establish a double reporter transgenic mice for monitoring Cre recombinase activity. Methods ZAP DNA fragment with lacZ and human alkaline phosphatase (hAP) gene was microinjected into the male pronucleus of 554 fertilized eggs from C57BL/6 mice. The founder mice and their progeny were screened for integration of transgene into the mouse genome by PCR and Southern blotting. The expression of lacZ transgene at early embryos from F1 generation mice was analyzed by X gal staining. Results A total of 398 survival ZAP DNA injected fertilized eggs were transfered to the oviducts of 21 pseudopregnant recipient mice. Of the 21 recipient mice, 13 became pregnancy and gave birth to 68 offspring mice. The zygote survival rate and birth rate were 71% (398/554) and 17% (68/398), respectively. Of the 68 offspring mice, 9 mice (5 males and 4 females) were identified by PCR and Southern blot analysis. Total integration rate and efficiency of transgene was 13% (9/68) and 1.6% (9/554), respectively. Nine mice as the founders were back crossed to set up F1 generation with other inbred C57BL/6 mice. Out of 9 transgenic mice, transmission of reporter gene in F1 offspring mice followed Mendelian rules, but the expression of lacZ protein was detected at the early embryonic stage (13.5 days postcoitum) in only 3 mice. Conclusion A double reporter transgenic mice for monitoring Cre recombinase activity is established.