RESUMO
Objective:To investigate thyroid function, physical growth, and psychological and behavioral development in children with congenital hypothyroidism.Methods:Thirty-two children with congenital hypothyroidism who were born in Yuyao People's Hospital from January 2014 to December 2018 were included in the observation group. Thirty healthy neonates who were born in the same period were included in the control group. Thyroid function index changes at the age of 1 year relative to at birth, physical, intellectual, and neuropsychological development and bone age at the age of 1 year were compared between the observation and control groups.Results:Thyroid-stimulating hormone level at birth was significantly higher in the observation group than in the control group [(18.23 ± 2.71) mU/L vs. (2.85 ± 0.34) mU/L, t = 30.84, P < 0.001]. Free thyroxine level at birth was significantly lower in the observation group than in the control group [(6.76 ± 1.54) pmol/L vs. (17.91 ± 2.04) pmol/L, t = 24.39, P < 0.001]. In the observation group, thyroid-stimulating hormone and free thyroxine levels at the age of 1 year were (2.68 ± 0.78) mU/L and (17.26 ± 2.11) pmol/L, respectively, which were not significantly different from those in the control group [(2.77 ± 0.63) mU/L and (17.54 ± 2.20) pmol/L, t = 0.50, 0.51, both P > 0.05]. Body weight, body length, head circumference, and bone age at the age of 1 year were (9.21 ± 1.20) kg, (79.84 ± 3.05) cm, (43.73 ± 1.42) cm, (1.01 ± 0.15) years old, respectively in the observation group, which were significantly lower than those in the control group [(10.12 ± 1.32) kg, (84.54 ± 3.41) cm, (45.85 ± 2.04) cm, (1.14 ± 0.28) years old, t = 2.84, 5.73, 4.77, 2.30, P < 0.05]. The proportion of children patients with bone age lag was significantly higher in the observation group than in the control group [21.88% (7/32) vs. 3.33% (1/30), χ2 = 4.74, P < 0.05]. There was a significant difference in intellectual development at the age of 1 year between the two groups ( χ2 = 7.05, P < 0.05). Gross movement, fine movement, adaptability, language ability, and social ability in the observation group were scored (90.43 ± 6.96) points, (92.03 ± 6.03) points, (88.45 ± 4.85) points, (84.04 ± 5.71) points, and (85.05 ± 6.17) points, respectively, which were significantly lower than those in the control group [(99.47 ± 5.40) points, (104.12 ± 5.71) points, (98.47 ± 5.22) points, (94.16 ± 4.98) points, and (104.34 ± 5.70) points ( t = 5.69, 8.09, 7.84, 7.42, 12.76, all P < 0.001]. Conclusion:Neonate patients with congenital hypothyroidism have obvious physical growth and psychological and behavioral development disorders. Early screening and treatment of neonatal congenital hypothyroidism should be strengthened to improve the prognosis.
RESUMO
In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.