Background and purpose:Immune checkpoint blockade(ICB)has become a promising strategy for treating cervical cancer(CC),but terminal T cell depletion still limits the therapeutic efficacy of ICB.The deletion of sorting nexin-9(SNX9)can inhibit thymocyte selection-associated high mobility group box protein(TOX),alleviate T cell exhaustion,and provide new ideas for preventing T cell exhaustion and enhancing the efficacy of cancer immunotherapy.Therefore,this study aimed to explore the immune escape mechanism mediated by the depletion of CD8+T cells induced by the SNX9/TOX signaling pathway in the CC microenvironment.Methods:Fifty-four peripheral blood samples were collected,including 18 from CC patients,18 from patients with high-grade squamous intraepithelial lesions(HSIL)and 18 from subjects with normal cervix.In addition,the study collected 153 pairs of CC and adjacent tissues from patients who received operation in our hospital for the first time.Clinicopathological features,tumor stages,follow-up records and other relevant clinicopathological data of CC patients were obtained from hospital records.The research was approved by the ethics committee of Changsha Fourth Hospital(approval number:20220206).A total of 24 mice were randomly assigned to the following four groups:immunoglobulin G(IgG)group,Anti-SNX9 group,Anti-programmed death-1(PD-1)group and Anti-SNX9+Anti-PD-1 group,with 6 mice in each group.Each group received intraperitoneal injection of blocking antibody and isotype control treatment respectively.ELISpot was used to detect the ability of CD8+T cells to secrete tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ).The expressions of TOX and SNX9 in cervical cancer tissues were detected by western blot and immunohistochemistry.Results:The expressions of SNX9 and TOX mRNA in peripheral blood mononuclear cell(PBMC)of CC patients were higher compared with HSIL and normal controls(P＜0.05).The positive cell level of SNX9 and TOX immunohistochemical score were higher in CC tissue than in adjacent tissues(t=18.63,21.10,P＜0.001).The high expression of SNX9 in CC was related to low differentiation/undifferentiation,tumor size,parauterine infiltration,vaginal infiltration,late FIGO stage and pelvic lymph node metastasis(P＜0.05).Compared with the low expression group of SNX9,the overall survival time of CC patients in the high expression group of SNX9 was shorter(P＜0.05).The percentage of CD8+SNX9+T cells was significantly higher in CC patients than in normal controls and HSIL patients(P＜0.05).The ability of CD8+SNX9+T cells to secrete cytokines(TNF-α and IFN-γ)was significantly lower compared with CD8+SNX9-T cells(P＜0.05).Compared with the Anti-SNX9 group,the growth and proliferation of cervical tumor,the expression of SNX9 and TOX protein in tumor tissue in the Anti-SNX9+Anti-PD-1 group further decreased(P＜0.05),and the level of infiltrating CD8+T lymphocytes in tumor tissue and the ability of CD8+T lymphocytes to secrete functional factors TNF-α and IFN-γ further increased(P＜0.05).Conclusion:SNX9/TOX signaling pathway exhibits enhanced activity in patients with cervical cancer and mouse models,and is related to immunosuppression.Targeting SNX9/TOX signaling pathway may be a potential therapeutic strategy for CC.

Objective:To establish and evaluate a second-tier screening method for neonatal congenital adrenal hyperplasia (CAH) and develop appropriate screening interpretation criteria.Methods:We employed liquid chromatography-tandem mass spectrometry to simultaneously detect five steroid hormones in dried blood spots: 17α-hydroxyprogesterone (17α-OHP), androstenedione (A4), 11-deoxycortisol (11-DOC), 21-deoxycortisol (21-DOC), and cortisol (F), calculating (17α-OHP+A4)/F and (17α-OHP+21-DOC)/F ratios for second-tier CAH screening. The study utilized 429 residual dried blood spot samples from neonates (0-7 days) who completed first-tier screening between January 2020 and March 2024 in Guangzhou Women and Children's Medical Center, Guangzhou Medical University, including first-tier negatives ( n=369), confirmed false positives ( n=50), and CYP21A2-confirmed 21-hydroxylase deficiency patients ( n=10). Mann-Whitney U and Kruskal-Wallis tests analyzed steroid concentration variations across gestational ages and birth weights in all negative samples, with reference intervals established via P2.5- P97.5 percentiles and screening cutoffs set at population P97.5. Receiver operating characteristic (ROC) curve analysis identified optimal interpretation indicators among steroid hormone profiles, with second-tier screening performance evaluated by comparing sensitivity and specificity across different steroid hormone indicators to establish the optimal diagnostic criteria. Results:The five steroid hormones demonstrated intra-assay precision with coefficient of variation (CV) of 9.8%-14.2% and inter-assay precision with CV of 4.7%-14.4% across three different concentration levels of quality control materials. Accuracy ranged from 98.5% to 110.0% and the lower limits of quantification were 0.25 ng/ml for 17α-OHP, 0.05 ng/ml for A4/11-DOC, 0.31 ng/ml for 21-DOC, and 0.1 ng/ml for F. Stratification by gestational age categorized 17α-OHP into ≤31, 32-34, and ≥35 weeks; A4 into ≤31, 32-36, and ≥37 weeks; and 11-DOC into ≤31 and ≥32 weeks, while the remaining indicators were not stratified. When grouped by birth weight (low/normal), all measured parameters except 21-DOC showed statistically significant differences between groups (all P<0.05). Established reference intervals included 17α-OHP: 0.53-7.82 ng/ml (≤31 weeks), <0.25-3.60 ng/ml (32-34 weeks), <0.25-1.64 ng/ml (≥35 weeks); A4: 0.12-2.36 ng/ml (≤31 weeks), <0.05-1.45 ng/ml (32-36 weeks), 0.17-0.95 ng/ml (≥37 weeks); 11-DOC: 0.43-4.04 ng/ml (≤31 weeks), 0.08-1.46 ng/ml (≥32 weeks); F: 1.70-83.70 ng/ml; 21-DOC: <0.31-0.69 ng/ml; (17α-OHP+A4)/F: 0.01-0.74; and (17α-OHP+21-DOC)/F: 0.01-0.69. Comprehensive comparison of CAH second-tier screening performance demonstrated that interpretation based on elevated 17α-OHP accompanied by either elevated 21-DOC or elevated ratios [(17α-OHP+A4)/F or (17α-OHP+21-DOC)/F] achieved 100% sensitivity, 96% specificity, and a 96% reduction in false-positive rate. Conclusion:The application of liquid chromatography-tandem mass spectrometry for multi-steroid hormone profiling in second-tier neonatal CAH screening, utilizing gestational age-specific 17α-OHP cutoffs combined with elevated 21-DOC or ratio criteria, demonstrated 100% screening sensitivity while substantially reducing false-positive rates from primary screening, though further validation with expanded sample sizes remains necessary.

Background and purpose:Immune checkpoint blockade(ICB)has become a promising strategy for treating cervical cancer(CC),but terminal T cell depletion still limits the therapeutic efficacy of ICB.The deletion of sorting nexin-9(SNX9)can inhibit thymocyte selection-associated high mobility group box protein(TOX),alleviate T cell exhaustion,and provide new ideas for preventing T cell exhaustion and enhancing the efficacy of cancer immunotherapy.Therefore,this study aimed to explore the immune escape mechanism mediated by the depletion of CD8+T cells induced by the SNX9/TOX signaling pathway in the CC microenvironment.Methods:Fifty-four peripheral blood samples were collected,including 18 from CC patients,18 from patients with high-grade squamous intraepithelial lesions(HSIL)and 18 from subjects with normal cervix.In addition,the study collected 153 pairs of CC and adjacent tissues from patients who received operation in our hospital for the first time.Clinicopathological features,tumor stages,follow-up records and other relevant clinicopathological data of CC patients were obtained from hospital records.The research was approved by the ethics committee of Changsha Fourth Hospital(approval number:20220206).A total of 24 mice were randomly assigned to the following four groups:immunoglobulin G(IgG)group,Anti-SNX9 group,Anti-programmed death-1(PD-1)group and Anti-SNX9+Anti-PD-1 group,with 6 mice in each group.Each group received intraperitoneal injection of blocking antibody and isotype control treatment respectively.ELISpot was used to detect the ability of CD8+T cells to secrete tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ).The expressions of TOX and SNX9 in cervical cancer tissues were detected by western blot and immunohistochemistry.Results:The expressions of SNX9 and TOX mRNA in peripheral blood mononuclear cell(PBMC)of CC patients were higher compared with HSIL and normal controls(P＜0.05).The positive cell level of SNX9 and TOX immunohistochemical score were higher in CC tissue than in adjacent tissues(t=18.63,21.10,P＜0.001).The high expression of SNX9 in CC was related to low differentiation/undifferentiation,tumor size,parauterine infiltration,vaginal infiltration,late FIGO stage and pelvic lymph node metastasis(P＜0.05).Compared with the low expression group of SNX9,the overall survival time of CC patients in the high expression group of SNX9 was shorter(P＜0.05).The percentage of CD8+SNX9+T cells was significantly higher in CC patients than in normal controls and HSIL patients(P＜0.05).The ability of CD8+SNX9+T cells to secrete cytokines(TNF-α and IFN-γ)was significantly lower compared with CD8+SNX9-T cells(P＜0.05).Compared with the Anti-SNX9 group,the growth and proliferation of cervical tumor,the expression of SNX9 and TOX protein in tumor tissue in the Anti-SNX9+Anti-PD-1 group further decreased(P＜0.05),and the level of infiltrating CD8+T lymphocytes in tumor tissue and the ability of CD8+T lymphocytes to secrete functional factors TNF-α and IFN-γ further increased(P＜0.05).Conclusion:SNX9/TOX signaling pathway exhibits enhanced activity in patients with cervical cancer and mouse models,and is related to immunosuppression.Targeting SNX9/TOX signaling pathway may be a potential therapeutic strategy for CC.

Objective:To establish and evaluate a second-tier screening method for neonatal congenital adrenal hyperplasia (CAH) and develop appropriate screening interpretation criteria.Methods:We employed liquid chromatography-tandem mass spectrometry to simultaneously detect five steroid hormones in dried blood spots: 17α-hydroxyprogesterone (17α-OHP), androstenedione (A4), 11-deoxycortisol (11-DOC), 21-deoxycortisol (21-DOC), and cortisol (F), calculating (17α-OHP+A4)/F and (17α-OHP+21-DOC)/F ratios for second-tier CAH screening. The study utilized 429 residual dried blood spot samples from neonates (0-7 days) who completed first-tier screening between January 2020 and March 2024 in Guangzhou Women and Children's Medical Center, Guangzhou Medical University, including first-tier negatives ( n=369), confirmed false positives ( n=50), and CYP21A2-confirmed 21-hydroxylase deficiency patients ( n=10). Mann-Whitney U and Kruskal-Wallis tests analyzed steroid concentration variations across gestational ages and birth weights in all negative samples, with reference intervals established via P2.5- P97.5 percentiles and screening cutoffs set at population P97.5. Receiver operating characteristic (ROC) curve analysis identified optimal interpretation indicators among steroid hormone profiles, with second-tier screening performance evaluated by comparing sensitivity and specificity across different steroid hormone indicators to establish the optimal diagnostic criteria. Results:The five steroid hormones demonstrated intra-assay precision with coefficient of variation (CV) of 9.8%-14.2% and inter-assay precision with CV of 4.7%-14.4% across three different concentration levels of quality control materials. Accuracy ranged from 98.5% to 110.0% and the lower limits of quantification were 0.25 ng/ml for 17α-OHP, 0.05 ng/ml for A4/11-DOC, 0.31 ng/ml for 21-DOC, and 0.1 ng/ml for F. Stratification by gestational age categorized 17α-OHP into ≤31, 32-34, and ≥35 weeks; A4 into ≤31, 32-36, and ≥37 weeks; and 11-DOC into ≤31 and ≥32 weeks, while the remaining indicators were not stratified. When grouped by birth weight (low/normal), all measured parameters except 21-DOC showed statistically significant differences between groups (all P<0.05). Established reference intervals included 17α-OHP: 0.53-7.82 ng/ml (≤31 weeks), <0.25-3.60 ng/ml (32-34 weeks), <0.25-1.64 ng/ml (≥35 weeks); A4: 0.12-2.36 ng/ml (≤31 weeks), <0.05-1.45 ng/ml (32-36 weeks), 0.17-0.95 ng/ml (≥37 weeks); 11-DOC: 0.43-4.04 ng/ml (≤31 weeks), 0.08-1.46 ng/ml (≥32 weeks); F: 1.70-83.70 ng/ml; 21-DOC: <0.31-0.69 ng/ml; (17α-OHP+A4)/F: 0.01-0.74; and (17α-OHP+21-DOC)/F: 0.01-0.69. Comprehensive comparison of CAH second-tier screening performance demonstrated that interpretation based on elevated 17α-OHP accompanied by either elevated 21-DOC or elevated ratios [(17α-OHP+A4)/F or (17α-OHP+21-DOC)/F] achieved 100% sensitivity, 96% specificity, and a 96% reduction in false-positive rate. Conclusion:The application of liquid chromatography-tandem mass spectrometry for multi-steroid hormone profiling in second-tier neonatal CAH screening, utilizing gestational age-specific 17α-OHP cutoffs combined with elevated 21-DOC or ratio criteria, demonstrated 100% screening sensitivity while substantially reducing false-positive rates from primary screening, though further validation with expanded sample sizes remains necessary.

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 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 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.