Objective:To conduct clinical analysis and genetic variant detection in patients with congenital hypothyroidism, and to explore the role of paired box 8(PAX8) gene variants in the pathogenic mechanism of congenital hypothyroidism, thereby providing insights for its diagnosis and management.Methods:This study was a retrospective analysis based on previously collected clinical and genetic data. Clinical information from 763 children with congenital hypothyroidism and some of their family members was collected between 2014 and 2025. Peripheral blood samples were obtained for genomic DNA extraction. Next-generation sequencing was performed to detect pathogenic variants in the patients. The identified pathogenic PAX8 variants were confirmed by Sanger sequencing and pedigree analysis. Functional analysis of the variant was then performed using bioinformatics tools.Results:Five PAX8 variants were identified among 763 children with congenital hypothyroidism. One proband was identified through neonatal screening and subsequently confirmed to have congenital hypothyroidism. Thyroid ultrasound revealed a small thyroid gland. A heterozygous missense variant in the PAX8 gene, NM_003466.3: c.91C>T: p.R31C, was identified in this proband, resulting in the substitution of arginine with cysteine at Codon 31 of the PAX8 protein. Sanger sequencing confirmed that this was a de novo variant, as it was absent in the proband′s parents. Multiple bioinformatic programs consistently predicted the variant to be deleterious. According to the American College of Medical Genetics and Genomics(ACMG) guidelines, the variant was classified as pathogenic. Conclusions:A de novo PAX8 variant, p. R31C, was identified in a patient with congenital hypothyroidism. This variant is located within the paired domain of the PAX8 protein and may impair the protein′s function by disrupting its binding to DNA.

Objective:To conduct clinical analysis and genetic variant detection in patients with congenital hypothyroidism, and to explore the role of paired box 8(PAX8) gene variants in the pathogenic mechanism of congenital hypothyroidism, thereby providing insights for its diagnosis and management.Methods:This study was a retrospective analysis based on previously collected clinical and genetic data. Clinical information from 763 children with congenital hypothyroidism and some of their family members was collected between 2014 and 2025. Peripheral blood samples were obtained for genomic DNA extraction. Next-generation sequencing was performed to detect pathogenic variants in the patients. The identified pathogenic PAX8 variants were confirmed by Sanger sequencing and pedigree analysis. Functional analysis of the variant was then performed using bioinformatics tools.Results:Five PAX8 variants were identified among 763 children with congenital hypothyroidism. One proband was identified through neonatal screening and subsequently confirmed to have congenital hypothyroidism. Thyroid ultrasound revealed a small thyroid gland. A heterozygous missense variant in the PAX8 gene, NM_003466.3: c.91C>T: p.R31C, was identified in this proband, resulting in the substitution of arginine with cysteine at Codon 31 of the PAX8 protein. Sanger sequencing confirmed that this was a de novo variant, as it was absent in the proband′s parents. Multiple bioinformatic programs consistently predicted the variant to be deleterious. According to the American College of Medical Genetics and Genomics(ACMG) guidelines, the variant was classified as pathogenic. Conclusions:A de novo PAX8 variant, p. R31C, was identified in a patient with congenital hypothyroidism. This variant is located within the paired domain of the PAX8 protein and may impair the protein′s function by disrupting its binding to DNA.

Objective:To assess clinical and genetic features in a patient with thyroid hormone resistance syndrome(RTH) and explore the pathogenic mechanism.Methods:The clinical data of the proband was collected. The genomic DNA was extracted from peripheral blood samples of the patients. The pathogenic variant was identified using whole-exome sequencing and confirmed by Sanger sequencing. Then the function of the mutation sites was detected by bioinformatics.Results:The patient presented with chest distress, palpitation, and persistent atrial fibrillation, along with elevated levels of serum free triiodothyronine(FT 3), free thyroxine(FT 4), and thyroid stimulating hormone(TSH), which suggested RTH clinically. The genetic analysis identified a heterozygous mutant of THRβ(c.1313G>A) gene at exon 8, which was a missense mutation causing the substitution of arginine to histidine at 438 position of the protein(p.R438H). Its inheritance pattern was unknown. This mutation was considered as a new one that had not been reported. Conclusion:A novel pathogenic THRβ gene mutation was found in the patient with RTH, which might be the cause of this disease. This variant c. 1313G>A is located in the ligand binding domain of THRβ, which might result in low protein activity.

Objective:This study aimed to identify mutations of the thyroglobulin(TG) gene and inheritance patterns in Chinese patients with congenital hypothyroidism.Methods:Clinical data of 235 children with congenital hypothyroidism and some of their families were collected from 2014 to 2018, and peripheral blood samples were collected for DNA extraction. Genomic DNA was extracted from the peripheral blood and TG gene was amplified with primers designed for each exon region and exon-intron junction region. Next generation sequencing technology and bioinformatics analysis were used to obtain TG gene mutations, followed by validation through Sanger sequencing. In addition, two compound heterozygotes with TG mutations and their parents were tested by Sanger sequencing.Results:Among 235 patients with congenital hypothyroidism, thirty-four cases(14.47%) carried TG gene variants, of which 6 cases(2.55%) carried compound heterozygous mutations. In addition, one of these TG gene variants was a hotspot mutation(T1620M was carried by four patients), nine were novel mutations(T601M, V2423I, R2308S, C2005R, C2264F, L987R, K1645Efs*49, F311Lfs*91, and T1416M).Conclusion:The mutation rate of the TG gene is higher in Chinese patients with congenital hypothyroidism, and two pedigrees indicated an autosomal recessive inheritance pattern.

Objective:To investigate methods of molecular diagnosis and clinical features of 46, XY disorders of sexual development(DSD).Methods:A total of 206 cases of 46, XY DSD patients, who visited the Shanghai Ninth People′s Hospital, Shanghai Jiaotong University School of Medicine, from July 2009 to June 2021, underwent AA chip based on multiplex PCR and probe-capture-targeted next-generation sequencing. Clinical features of patients with genetic diagnosis were analyzed.Results:Among 206 patients, the diagnostic rate of patients with micropenis, hypospadias and cryptorchidism was the highest, up to 75.28%. Almost all patients had different degrees of undermasculinized external genitalia. The most frequent phenotype was micropenis with hypospadias(87.25%). Only one gene variant was detected in 81 patients(39.32%), multiple genetic variants were detected in 104 patients(50.49%), and no gene variant was identified in 21 patients(10.19%). 107 patients had definite genetic diagnosis, with a diagnostic rate of 51.94% by adding the pathogenic and likely pathogenic ratios following the American College of Medical Genetics and Genomics(ACMG) guidelines, including 40 patients of steroid 5α-reductase type 2(SRD5A2) variants(37.38%), 36 patients of androgen receptor(AR) variants(33.64%), 13 patients of steroidogenic factor 1(NR5A1) variants(16.82%), 6 patients of 17β-hydroxysteroid dehydrogenases 3(HSD17B3) variants(5.61%), 2 patients of 17α-hydroxylase/17, 20-lyase enzyme(CYP17A1), Wilms′ tumor 1(WT1) and GATA binding protein 4(GATA4) variants(1.87%), and one patient of luteinizing hormone receptor(LHCGR) variant(0.93%). Gynecomastia was found in 29 of 81 postpubertal patients, of which 25(86.21%) had AR variants.Conclusions:46, XY DSD presents complex clinical manifestations and molecular etiologies. Targeted nextgeneration sequencing has the advantages of high throughput, high efficiency and low cost, which has a high value especially in etiological diagnosis of 46, XY DSD with large genetic heterogeneity.

Objective:Clinical and genetic analysis were conducted in 2 patients with hypophosphatasia(HPP) and their families to explore the pathogenic mechanism of HPP.Methods:The genomic DNA was extracted from peripheral blood of two patients with HPP and their family members. Sanger sequencing and pedigree verification were performed on the pathogenic variants identified using whole-exome sequencing. Then the function of the mutation sites was analyzed with bioinformatics software.Results:Proband 1 presented with developmental retardation, pectus funnel and premature loss of deciduous tooth, of which the serum alkaline phosphatase level was slightly lower than the bound of the normal range. Two complex heterozygous missense variants c. 1120G>A and c. 1334C>G of ALPL gene were detected in the proband 1 which were inherited from his parents respectively, showing an autosomal recessive inheritance. Both the variants were predicted to inflict deleterious effects on ALPL gene function by multiple bioinformatics program, and were classified as likely pathogenetic variants according to American College of Medical Genetics and Genomics(ACMG) guidelines. Proband 2 showed three missing permanent teeth and the significantly lower level of serum alkaline phosphatase than normal range. A heterozygous variant c. 1190-3C>G of ALPL gene was detected in proband 2 whose pattern of inheritance was unknown. The clinical significance of this variant was unknown according to ACMG standards and guidelines. All of these variants were considered as novel since none of them has been reported. Along with the above combined results, proband 1 and 2 were diagnosed as childhood HPP and Odontohypophosphatasia, respectively.Conclusion:This study reinforced the relationship between HPP and variants in ALPL gene. Two variants, c. 1120G>A and c. 1334C>G, were located in the homodimer interface and crown domain of tissue-nonspecific alkaline phosphatase(TNSALP), respectively, while c. 1190-3C>G were located in the splice sites, which might result in low TNSALP activity.

Objective:To investigate the clinical features and treatment outcome of Kallmann syndrome(KS) caused by fibroblast growth factor receptor-1(FGFR1) gene mutation in 4 patients.Methods:Targeted next-generation sequencing(NGS) was performed on thirty KS and normosmic idiopathic hypogonadotropic hypogonadism(nIHH) patients. FGFR1 mutation was identified in four KS patients. The clinical data, laboratory and imaging examinations, and treatment outcome were retrospectively analyzed.Results:Four male patients, aging from 11 to 22 years old, presented as micropenis, and with olfactory dysfunction. Among them, two had history of cryptorchidism, three had history of cleft lip and palate repair surgery. The most severe patient presented with short stature, left microtia and dental agenesis. FGFR1 heterozygous mutation was identified in all four patients, two were point mutation(p.Y374X; p. E670K), and the other was frameshift mutation(p.S346Yfs*61; p.S723*fs*1). One patient, who started treatment of the pulsatile GnRH pump during his youth, succeeded in having two babies.Conclusion:Patients with Kallmann syndrome caused by FGFR1 mutation presents complex clinical manifestations. Besides dysosmia, micropenis, microrchidia, and delayed pubertal development are the main clinical manifestations in male patients. Symptoms such as cleft lip and palate are helpful for early recognition. Genotyping analysis is crucial to confirm the diagnosis. The pulsatile GnRH pump can produce satisfactory therapeutic effect, but the age of initiating therapy should be carefully considered.

Objective To investigate the prevalence of DUOX2 mutations in Chinese patients with congenital hypothyroidism (CH) and to discuss the inheritance pattern of DUOX2 gene.Methods Blood samples were collected from 91 CH children and their genomic DNA was extracted from peripheral blood leukocytes.All exons and exon-intron boundaries of DUOX2 were analyzed by target next-generation sequencing and family trios was established to study the inheritance pattern of DUOX2 gene.Results Fifty-four out of 91 children with CH carried DUOX2 mutation, with a prevalence of 59.34%.Of the 54 CH children, 36 carried DUOX2 biallelic mutations.In all 12 family trios with probands carrying biallelic DUOX2 mutations, the parents carried heterozygous DUOX2 mutations while still showing normal thyroid function, suggesting that CH caused by DUOX2 mutations is inherited in an autosomal recessive manner.Conclusion DUOX2 gene is one of the most frequently mutated genes in Chinese CH patients and its inheritance pattern is an autosomal recessive one.

Objective To identify the association of thyroid stimulating hormone receptor ( TSHR ) gene intron 1 susceptible loci and 4p14 susceptible locus rs6832151 polymorphisms with Graves’ disease ( GD) in Han Chinese population in Bengbu, Anhui, China. The gene-gene interaction among TSHR intron 1 susceptible loci and 4p14 susceptible locus rs6832151 was also investigated. Methods The genotypes of the single-nucleotide polymorphisms ( SNPs) were analyzed by Taqman probe technique on Fluidigm EP1 platform in 611 patients with GD and 555 control subjects, and linkage analysis, correlation analysis, haplotype analysis, and epistasis analysis with them were performed. Results Six SNPs in two candidate genes(rs12101261, rs4903964,rs179247, rs2284722 and rs17111394 in TSHR, rs6832151 in 4p14) were associated with GD (all P<0. 05). The frequency distributions of haplotypes of SNPs in TSHR intron 1 ( AGTA, GGCG, AATA, and CC) were significantly different between GD and control groups(all P<0. 01). There existed the interactions between rs179247 and rs12101261 in TSHR(P=0. 001) and among rs179247(TSHR),rs4903964(TSHR) and rs6832151(4p14) (P=0. 001). Conclusions rs683215 in 14p14 and rs12101261, rs4903964, rs179247, rs2284722 and rs17111394 in TSHR intron 1 were susceptible loci of GD in the Chinese Han population from Bengbu. The haplotypes in TSHR intron 1 were associated with GD. There exists the interaction between the SNPs in TSHR and 4p14,which may change the risk of GD.

[Summary] The activities of 17α-hydroxylase and 17,20 lyase of cytochrome P450c17 were evaluated by ELISA in NCI-H295R cells after treatment with palmitate and oleate. The results showed that 0. 75 mmol/L plamitate did not influence the activity of 17α-hydroxylase, but increased the activity of 17, 20 lyase by 74. 3% ( P<0. 05). Oleate at the same concentration did not change the activity of 17,20 lyase. There were no significant changes in the protein expressions of P450c17, P450 oxidoreductase, and cytochrome b5 after treatment with palmitate and oleate. However,reactive oxygen species in cells were elevated by palmitate. The results suggest that exposure to palmitate may increase androgen production by inducing 17, 20 lyase activity of P450c17 in NCI-H295R cells, which is related with oxidative stress-mediated post-translational regulation of the enzyme.