Objectives:To explore the GAA varient spectrum and the genotype-phenotype correlations in patients with glycogen storage disease type Ⅱ (Pompe disease, PD), as well as to estimate the disease incidence based on carrier rate of GAA varients in Guangzhou population.Methods:A total of 57 PD cases were retrospectively enrolled at Guangzhou Women and Children′s Medical Center from January 1, 2010 to May 31, 2020. All patients presented symptoms before the age of 18 years. Each diagnosis was further confirmed by GAA enzyme activity and GAA variants. The carrier rate of GAA varients was calculated based on variants detected by whole exon sequencing among 2 395 healthy children in Guangzhou.Results:Among the 57 PD patients (including male 26, female 31),twenty-eight patients with infantile onset PD (IOPD) presented with progressive general muscle weakness and cardiomyopathy. The mean ages of symptom onset and diagnosis were (2.5±1.4) and (5.0±3.0) months, respectively. Twenty-six cases died in the first year after birth.Twenty-three patients with late onset PD (LOPD) presented with progressive muscle weakness. Seven of them had respiratory failure at diagnosis. The mean ages of symptom onset and diagnosis were (12.0±5.0) and (17.0±7.5) years, respectively. Six children with atypical IOPD showed motor delay, muscle weakness and cardiomyopathy. Their diagnosis was confirmed at 2.5-7.0 years of age. Among the 57 patients, 47 different variants were identified in the GAA gene. Three variants: c.797C>T, c.1109G>A and c.1757C>T were novel. c.1935C>A (25/114, 21.9%) and c.2238G>C (15/114, 13.2%) were the most common variants, detected in 57.1% of IOPD and 65.2% (15/23) of LOPD patients, respectively. Among the 28 IOPD patients, 26 cases (92.9%) carried at least one missense variant which indicated positive cross-reactive immunologic material (CRIM). The carrier rate of pathogenic variants in GAA gene among healthy children was 24/2 395. The estimated incidence of PD in this population is about 1/40 000. The frequencies of pseudodeficiency variants c.1726G>A and c.2065G>A homozygotes were 26.3% (15/57) and 35.1% (20/57) in PD patients, which were significantly higher than those (1.7% (40/2 395) and 3.9% (94/2 395)) in healthy children (χ2=151.2, 121.9; both P<0.01). Conclusions:PD presents as a spectrum, some as atypical IOPD. The c.1935C>A and c.2238G>C are common variants, correlated with IOPD and LOPD respectively. The c.796C>T and c.1082C>T are usually found in atypical IOPD. The majority of IOPD patients is predicted to be CRIM positive. The estimated incidence of PD is about 1/40 000.

Objective:To investigate the sensitivity of newborn screening for neonatal intrahepatic cholestasis caused by Citrin deficiency (NICCD) based on tandem mass spectrometry and the carrying rate of known pathogenic variants of SLC25A13 in Guangzhou population. Methods:A total of 124 250 neonates born in Guangzhou from January 1, 2015 to December 31, 2018 were performed newborn screening for NICCD by tandem mass spectrometry technology. SLC25 A13 gene mutation analysis was performed to diagnose patients with suspected NICCD.The carrying rate of known pathogenic variants of the SLC25 A13 gene in the whole exon sequencing results of 2 395 healthy children in Guangzhou was retrospective analyzed. Results:Among the 124 250 screened neonates, 31 cases were screened positive for NICCD and one of them was confirmed.Three false negative patients with NICCD were found in this cohort.NICCD screening sensitivity was 25%(1/4 cases). All of the four patients were homozygous for c. 851_854del of SLC25A13.Among 2 395 controls, 60 cases were detected heterozygous variant of SLC25A13, including 8 kinds of reported pathogenic variants.The carrying rate of pathogenic alleles was 1/40 (60/2 395 cases). The estimated prevalence of citrin deficiency was about 1/6 400.The most common variant was c. 851_854del (56.7%, 34/60 cases), and the second was c. 790G>A (23.3%, 14/60 cases). The controversial variant c. 2T>C was detected in 113 children with heterozygous and 2 cases with homozygous and the carrying rate of c. 2T>C was 1/20(117/2 395 cases). Conclusions:The carrying rate of pathogenic variants of SLC25A13 and the estimated prevalence of Citrin deficiency in Guangzhou population are high.The sensitivity of newborn screening for NICCD by tandem mass spectrometry is limited.Even if the negative results for screening of multiple genetic and metabolic diseases by tandem mass spectrometry, it is recommended to recheck blood for newborns or infants with delayed jaundice to avoid missed diagnosis.

Objective:To reveal the relationship between G6PD genotypes and the G6PD enzyme activities in dried blood spots of newborn screening.Methods:Simple random sampling procedure was used in this study. The fluorescence PCR melting curve analysis was performed to classify G6PD gene variants in 635 neonates coming from Guangzhou Newborn Screening Center during October 1 to 20, 2016, including 15 reported variants. Those samples consisted of 377 cases with screening positive results (261 from males and 116 from females) and 258 cases with screening negative results (32 from males and 226 from females). The cut-off value of G6PD was less than 2.6 U/g Hb in dry blood spots. Sanger sequencing for G6PD gene was used in 7 cases with screening negative results under simple random sampling. One-way ANOVA and least significant difference method (LSD) test were performed to compare the difference of G6PD activity among genotypes.Results:The top 6 frequency of G6PD gene variants were c.1388G>A(35.07%), c.1376G>T(32.13%), c.95A>G(12.72%), c.871G>A(8.32%), c.1024C>T(4.08%) and c.392G>T(2.28%), accounting for 94.62% of all variant alleles (580/613). A total of 253 males positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 98.06%(253/258). The mean values of G6PD activities for c.1376G>T,c.95A>G and c.1388G>A were 0.85, 1.10 and 1.28 U/g Hb, respectively. There were significant differences among the three groups ( F=28.7, P<0.01). A total of 105 females positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 90.52%(105/116). The positive rate of G6PD enzyme activity was 26.95% among 256 females with one point mutation while it was 83.72% in females with multi-allele variants. The G6PD activity of heterozygous females was (2.9±0.8) U/g Hb, which was significant higher than that of females with multi-allele variants (1.5±1.0) U/g Hb ( t=8.6, P<0.01). Conclusions:G6PD activities in dried blood spots were related to G6PD genotypes in males. They were also associated with the numbers of allele variants in females. Newborn screening for G6PD deficiency can be used to detect most of G6PD-deficient hemizygotes and female patients with multi-allele variants, which is helpful for preventing neonatal jaundice and medicine application.

Objective:To evaluate and improve the performance of the newborn screening program for primary carnitine deficiency (PCD) based on tandem mass spectrometry and to investigate the incidence of PCD and molecular characteristics of SLC22A5 gene in Guangzhou.Methods:A total of 200 180 neonates born in Guangzhou from 2015 to 2019 were enrolled into the newborn screening program for PCD by tandem mass spectrometry at Guangzhou Newborn Screening Center. The positive results of screening for PCD was defined as free carnitine (C0) less than 10 μmol/L with decreased acylcarnitine species in dried blood spots of three to seven days after birth. Screen-positive newborns and their mothers were recalled for another blood spot sample. The diagnosis was confirmed based on decreased levels of C0 and acylcarnitine species in recalled blood spots and genetic analysis in SLC22A5 gene sequencing. The utility of using the sum of propionylcarnitine and palmitoylcarnitine (C3+C16) as a biomarker for acylcarnitine species in newborn screening was retrospectively evaluated. The levels of C0 and (C3+C16) at first screening were compared between newborns with PCD and newborns born to mothers with PCD by independent t test. The variant spectrum and known pathogenic variants carrier rate of SLC22A5 in 2 395 healthy children in Guangzhou Women and Children′s Medical Center through whole exon sequencing were analyzed. Results:Among 200 180 neonates, 239 (0.12%) cases were screen-positive for PCD. A total of 37 patients including 15 newborns and 22 mothers had confirmed PCD. The incidence of PCD was 1/13 345 in newborns and 1/9 099 in mothers, respectively. The positive predictive value of this program was 15.5%. Taking cutoff values of C0<8.5 μmol/L or C0 8.5~9.9 μmol/L with (C3+C16)<2 μmol/L, the number of screen-positive cases would be reduced from 810 to 224 without additional false negative case, when compared with cutoff value C0<10 μmol/L only. Both levels of C0 and (C3+C16) at first screening were not significant difference between newborns with PCD and newborns born to mothers with PCD ((6.2±2.4) vs. (5.0±1.8) μmol/L, (1.4±0.4) vs. (1.2±0.5) μmol/L, t=3.826, 0.326; P=0.058, 0.572). Seven PCD mothers experienced moderate fatigue and dizziness in the morning. One of them presented with cardiomyopathy in pregnancy. Genetic analysis of the SLC22A5 gene showed that p.S467C, p.F17L, p.R254X were the three most common variants in newborns with PCD. In PCD mothers and healthy children, the p.S467C, p.F17L and R399W were the three most common whereas the severe variant p.R254X was rare. The population carrier rate for pathogenic variants was 1 in 65 and the estimated incidence of PCD was about 1/16 500. Conclusions:Newborn screening can detect PCD both in newborns and mothers. Adding a quantitative biomarker (C3+C16) <2 μmol/L into the newborn screening program can improve the PCD screen performance. The severe variant p.R253X was common in PCD newborns but rare in PCD mothers and healthy children, indicating that the current screening program maybe failed to detect all PCD newborns and under-estimated the incidence rate of PCD in Guangzhou.

Objective:To reveal the relationship between G6PD genotypes and the G6PD enzyme activities in dried blood spots of newborn screening.Methods:Simple random sampling procedure was used in this study. The fluorescence PCR melting curve analysis was performed to classify G6PD gene variants in 635 neonates coming from Guangzhou Newborn Screening Center during October 1 to 20, 2016, including 15 reported variants. Those samples consisted of 377 cases with screening positive results (261 from males and 116 from females) and 258 cases with screening negative results (32 from males and 226 from females). The cut-off value of G6PD was less than 2.6 U/g Hb in dry blood spots. Sanger sequencing for G6PD gene was used in 7 cases with screening negative results under simple random sampling. One-way ANOVA and least significant difference method (LSD) test were performed to compare the difference of G6PD activity among genotypes.Results:The top 6 frequency of G6PD gene variants were c.1388G>A(35.07%), c.1376G>T(32.13%), c.95A>G(12.72%), c.871G>A(8.32%), c.1024C>T(4.08%) and c.392G>T(2.28%), accounting for 94.62% of all variant alleles (580/613). A total of 253 males positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 98.06%(253/258). The mean values of G6PD activities for c.1376G>T,c.95A>G and c.1388G>A were 0.85, 1.10 and 1.28 U/g Hb, respectively. There were significant differences among the three groups ( F=28.7, P<0.01). A total of 105 females positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 90.52%(105/116). The positive rate of G6PD enzyme activity was 26.95% among 256 females with one point mutation while it was 83.72% in females with multi-allele variants. The G6PD activity of heterozygous females was (2.9±0.8) U/g Hb, which was significant higher than that of females with multi-allele variants (1.5±1.0) U/g Hb ( t=8.6, P<0.01). Conclusions:G6PD activities in dried blood spots were related to G6PD genotypes in males. They were also associated with the numbers of allele variants in females. Newborn screening for G6PD deficiency can be used to detect most of G6PD-deficient hemizygotes and female patients with multi-allele variants, which is helpful for preventing neonatal jaundice and medicine application.

Objective: To investigate prospectively molecular and clinical characteristics of infants with congenital hypothyroidism (CH) caused by DUOX2 mutations in Guangzhou. Methods: A population-based cohort of 83 patients with CH were recruited based on newborn screening results among 108 899 newborns who were born in Guangzhou between April 1 and September 30 in 2015.Genetic analysis of DUOX2 hotspots(including 11 exons)by PCR-direct sequencing was performed in 52 patients with suspected thyroid dyshormonogenesis (SDH) according to thyroid ultrasound at diagnosis.All the patients were followed up for 3 years.The data of this cohort study(prevalence of CH, detection rate of DUOX2, clinical features) were compared with those of 96 patients with SDH in 2011-2012. Results: (1) The incidence of CH in 2015 was 1∶1 312, and 73.5%(61/83 cases) of CH patients were classified as SDH.Compared with those founded in 2011-2012, the incidence of CH was increased (1∶1 312 vs.1∶2 779), and the difference was significant (P<0.001), while the frequency of SDH was not different significantly (73.5%vs.76.6%, P=0.593). (2)There were 27 cases(51.9%) with SDH detected DUOX2 hotspots variants, including 6 cases with biallelic variants, 21 cases with monoallelic variants, and 1 possible new pathogenic variant p. S1091F.The p. K530X was the most common mutation accounting for 51.5%(17/33 cases) detected allelic and involving in 16 cases (30.8%) with SDH.Novel p. S1091F variant was probably damaging variant.Both detected rate and spectrum of DUOX2 variant were not significantly different compared with those in 2011-2012 (all P>0.05). (3) There were no significant differences in the levels of thyrotropin (bsTSH), serum TSH (sTSH), free thyroxine (FT4) and thyroglobulin in neonates with dry blood spot at diagnosis between children with DUOX2 and without DUOX2 variants cases(all P>0.05). Among 27 cases, 24(88.9%) patients with DUOX2 mutation were transient CH, and 3 cases were permanent CH. Conclusions The incidence of CH was increased in last few years in Guangzhou.Most of them were SDH, and 51.9% of SDH cases had DUOX2 hotspots variants.Temporary CH is the main clinical outcome.The p. K530X was the most common mutation in this cohort population.

Objective: To investigate the profiles of blood amino acid and acylcarnitine in early neonates with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and the sensitivity of newborn screening, and to explore potential biochemical metabolic markers for newborn screening program. Methods: Amino acid and acylcarnitine profiles in dried blood spots of newborn screening program were analyzed by tandem mass spectrometry (MS/MS). A total of 158 651 neonates born in Guangzhou from January 1, 2015 to June 30, 2019 were enrolled in this newborn screening program, and additionally 55 patients with NICCD confirmed by SLC25A13 gene analysis in Guangzhou Women and Children Medical Center were included in this study. NICCD screen-positive was defined as the cutoff value of citrulline (Cit) ≥ 30 μmol/L. The values of blood sampling time of the true positive group and those of the false negative group were compared by t-test. The levels of amino acid and acylcarnitine among different groups, including true positive group (Cit≥30 μmol/L), false negative group (Cit 21-<30 μmol/L and Cit<21 μmol/L) and the normal control group, were analyzed by F test, respectively. Results: Among 158 651 neonates, 39 neonates were positive for NICCD screening. Three of them were confirmed NICCD and 4 cases were found to be false negatives. The positive predictive value was 7.7% and the sensitivity was about 43.0%. Among 55 patients with NICCD, 18 cases (18/55, 32.7%) were true positives and 37 cases (37/55, 67.3%) were false negatives based on the cutoff value of citrulline in the dried blood spots for newborn screening. The blood sampling time was significantly different between true positive group and false negative group ((4.28±1.6) vs. (2.98±0.74) d, t=4.06, P<0.01). The increased levels of tyrosine((176.0±98.4) μmol/L), methionine ((37.0±26.9) μmol/L) and phenylalanine ((133.0±80.9)μmol/L) in Cit≥30 μmol/L group (n=18) were significantly different as compared with those in the other three groups, respectively (F=117.0, 58.5, 135.0, P<0.01). The levels of arginine ( (10.0±9.2) , (11.0±9.3) , (9.0±17.8) μmol/L), valine ( (119.0±29.8) , (107.6±14.1) , (102±68) μmol/L) and leucine ( (167.0±37.1) , (161.0±37.7) , (163.5±180.6) μmol/L) were not statistically significant among groups of Cit≥30 μmol/L(n=18), Cit21-<30 μmol/L(n=7) and Cit<21μmol/L(n=30,P>0.05), but they were significantly higher than those of the normal control group ((4±3), (78±21), (114.0±31.5) μmol/L, n=1 000), respectively(F=30.1, 23.0, 29.8, P<0.01). Alanine (Ala) ( (150±50) , (156.0±30.2), (168±105), (152±52) μmol/L) levels showed no significant difference (F=0.86, P>0.05) but the ratios of Ala/Cit (1.52±1.44, 6.82±1.56, 12.06±7.71, 19.42±6.27) decreased significantly among the four groups (F=69.0, P<0.05). The acylcarnitine levels showed no statistically significant results among the different groups (P>0.05). With Cit≥30 μmol/L and Ala/Cit<7.5 as cutoff values, the number of screen-positive cases reduced from 39 to 22 cases with no additional false negative case. With Cit≥21 μmol/L and Ala/Cit<7.5 as cutoff values the number of screen-positive cases increased to 117 cases with 1 additional true positive. Conclusions The profiles of blood amino acid in early neonates with NICCD present the increased levels of multiple amino acids including citrulline, tyrosine, methionine and phenylalanine, and decreased ratio of Ala/Cit. Taking citrulline and ratio of Ala/Cit as screening markers can improve the positive predictive value appropriately. The limited sensitivity of NICCD newborn screening may be related to early blood sampling time.

OBJECTIVETo observe the effects of acupuncture at opposite acupoints on expression of hepatocyte growth factor (HGF) in rats with skeletal muscle contusion, and to explore the mechanism of acupuncture at opposite acupoints on skeletal muscle contusion.

METHODSFifty-four Sprague Dawley (SD) rats were randomly divided into a blank group (6 rats), a model group (24 rats) and an opposing needling group (24 rats). The model group and opposing needling group were further divided into 1-day subgroup, 3-day subgroup, 5-day subgroup and 7-day subgroup, 6 rats in each one. No intervention was given in the blank group, while the model of skeletal muscle contusion was established in the model group and opposing needling group by self-made contusion device. 24 hours after contusion, electroacupuncture (EA) was applied at "Zusanli" (ST 36) and the corresponding points ofpoints at health side for 15 min, once a day. The subgroups of opposing needling group were treated for 1 day, 3 days, 5 days and 7 days, respectively. No treatment was given in the model group. Samples were collected in the subgroups 1 day, 3 days, 5 days and 7 days after treatment. The morphological change of injured gastrocnemius muscle was observed by using microscope after HE staining. The positive cell rate of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. The expression levels of HGF protein and PCNA protein were observed by Western blot.

RESULTS① The results of HE staining showed that, 1 day after contusion, the inflammatory cells of gastrocnemius muscle in the opposing needling group were less than those in the model group; 3 days and 5 days after contusion, myoblasts and myotubes in the opposing needling group were more than those in the model group; 7 days after contusion, the neonatal muscle cells in the opposing needling group were more than those in the model group. ② The results of immunohistochemistry showed that, 1 day, 3 days and 5 days after contusion, the positive cell rate of PCNA in the opposing needling group was significantly higher than that in the model group (all<0.001); 7 days after contusion, the positive cell rate of PCNA in the opposing needling group was significantly less than that in the model group (<0.001). ③ The results of Western blot showed that, 1 day, 3 days and 5 days after contusion, the expression of HGF protein and PCNA protein in the opposing needling group was significantly higher than that in the model group (all<0.05); 7 days after contusion, the expression of HGF protein and PCNA protein in the opposing needling group was significantly lower than that in the model group (all<0.05).

CONCLUSIONAcupuncture at opposite acupoints could regulate the expression of HGF and promote the activation, proliferation, migration and differentiation of muscle satellite cells in rats with skeletal muscle contusion, which could speed up the process of skeletal muscle injury repair.

Aiming at the new problems in orthodontics teaching and employment,this study aims to explore a more suitable undergraduate teaching mode of Orthodontics,and reform the teaching of Orthodontics for five-year program students in department of stomatology of Xi'an Medical University.During the teaching of Orthodontics,teachers use the aesthetic principles that students have learned to integrate into the orthodontics teaching,and guide students to analyze,understand and master orthodontic knowledge based on the principles of aesthetics.After the course,students give good feedback on the teaching reform,and their scores of basic knowledge and case analysis of Orthodontics theoretical examination have been improved.This teaching reform is very helpful for students to learn,understand and master the knowledge of orthodontics.It helps to improve classroom teaching effect,improve students' interest in learning,and adapt to the needs of discipline and industry development,so it is worth studying and promoting.

Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PD<1 700 U/L or G6PD/6PGD<1 in red blood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PD< 2.6 U/g Hb , a total of 687(4.2%) newborns showed positive screening results, including 560 (6.4%) males and 127(1.7%) females. (2) Among the newborns with positive screening results, 214 males and 122 females were randomly chosen for G6PD gene analysis. The results showed that 197 (92.1%) males were hemizygote and 108(88.6%) females were mutation carriers with one to four alleles. Among the newborns with negative screening results, 41 males with G6PD 2.6-2.8 U/g Hb and 436 females with G6PD 2.6-4.5 U/g Hb were chosen for genetic analysis.Mutations were detected in 5(12.2%)boys, and 226(51.8%) girls were carriers.G6PD gene Sanger sequency of seven highly suspicious patients showed that c.406C>T, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PD<2.6 U/g Hb yielded a positive predict value(PPV) of 93.5%, a false-positive rate of 0.5%, and a sensitivity of 99.0% for males. A PPV of 88.5%, a false-positive rate of 0.2% . The prevalence of severe type G6PD deficiency in females was about 1.5%. Compared with to genetic analysis, the sensitivity and PPV of G6PD activity assay in red blood cells were 95.5%, 97.2%, respectively. Conclusions The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will improve the accuracy of diagnosis of G6PD deficiency and detect more heterozygous females.