A total of 117 Yunnan-origin CH patients treated at the First People’s Hospital of Yunnan Province from January 2016 to January 2019 were retrospectively analyzed. These CH patients were diagnosed at an age ranging from 15 to 30 days; 54 males and 63 females were included. The inclusion criteria were as follows: (i) infants with negative thyroid autoantibody test results; (ii) lacking other congenital diseases or chromosomal abnormalities; and (iii) sporadic and non-consanguineous cases. The exclusion criteria were as follows: (i) central hypothyroidism or related syndromes and (ii) autoimmune diseases or family history thereof. The study area (Yunnan Province) has been classified as an iodine-sufficient region following implementation of the national universal salt iodization program.

This study was reviewed and approved by the Medical Ethics Committee of the First People’s Hospital of Yunnan Province (No. KHLL2023-KY037). Signed informed consent was obtained from the parents of all patients. The research protocol complied with the seventh revision of the Helsinki Declaration (2013).

The newborns were all screened at the Yunnan Province Newborn Screening Center. Within 72 hours to 20 days after birth, dried blood spot samples were collected from the heel of the newborns. These samples were then transported to the screening center laboratory through a cold chain system, and testing was completed within 3 days. TSH values were measured using a time-resolved fluorescence immunoassay (Perkin Elmer), with a cutoff value set at ≥8 μmol/L. Newborns with TSH screening results exceeding this positive cutoff value were recalled for serum thyroid function tests. Thyroid function was assessed in the patient’s serum using five different parameters, and values were compared to established normal reference ranges (TSH: 0.87∼6.15 mIU/L, T4: 77.8∼170.0 nmol/L, FT4: 12.0∼18.6 pmol/L, T3: 1.80∼3.68 nmol/L, FT3: 5.1∼8.0 pmol/L). A diagnosis of CH was made when the patient’s serum FT4 level fell below the minimum threshold and the TSH level exceeded the maximum detectable threshold.

The long diameter (L), wide diameter (W), and thick diameter (T) of the thyroid were measured using a SIEMENS-ACUSON-S2000 color Doppler ultrasound instrument. The probe frequency was set at 9~18 MHz, and the color scale parameter was conventionally set at 7 cm/s. The ultrasound examination procedure was as follows. After the child was calm or asleep, the child was placed in the supine position with appropriate neck elevation to fully expose the neck. The ultrasound probe was positioned in front of the neck, and during the examination, it was placed just below the thyroid cartilage. Transverse and longitudinal scans using two-dimensional ultrasound were initially conducted to carefully observe the thyroid’s size, shape, capsule, and internal echogenicity. The largest diameter of the right and left thyroid lobes and the isthmus was measured, and the formula V = 0.479LWT/1000 was used to calculate the volume of both lobes (15). Color Doppler flow imaging (CDFI) was used to assess the degree and distribution of blood flow within the thyroid. By comparing the thyroid volume of the CH patients with that of healthy infants of corresponding ages, the thyroids of the CH patients were categorized into normal, dysgenesis (agenesis or athyreosis, ectopy, orthotopic hypoplasia and hemiagenesis) and goiter groups.

Five milliliters of peripheral blood was collected from the patients and their parents. Genomic DNA (gDNA) was extracted according to the kit manufacturer’s standard procedure (MagPure Buffy Coat DNA Midi KF Kit, MAGEN BIOTECH). The gDNA was broken into 100–500 bp fragments by Shearing Enzyme Premix Reagent (ENZYMATICS), and 200–300 bp fragments were subsequently collected by magnetic beads (Vazyme DNA Clean Beads, VAZYME). An “A” base was added at the 3’ overhang after the end of the repair process to ensure that the fragments could pair with the “T” base with a special adapter, after which a single individual DNA library was constructed after Linker-mediated-PCR and purification. The library was enriched for 16 h at 65 °C by array hybridization (KAPA Hyper Exome, ROCHE), followed by elution and post-capture amplification. The products were subsequently subjected to analysis with a CaliperGX_1000 Kit (Caliper Life Sciences) and BMG to estimate the magnitude of enrichment. The qualified products were pooled and quantified according to different library sizes. Then, single strands of the library products were prepared for circularization, and DNB was generated. Finally, the products were sequenced with PE100 + 10 using an MGISEQ-2000.

To detect potential variants in the patients, bioinformatics processing and data analysis were performed after receiving the primary sequencing data. Comprehensive analysis was conducted to screen for mutations in 27 genes associated with CH, including HESX1, LHX3, LHX4, SOX3, OTX2, PROP1, POU1F1, TRHR, TSHB, LEPR, TPO, TSHR, TBL1X, IYD, PAX8, NKX2-1, FOXE1, IGSF1, NKX2-5, JAG1, GLIS3, CDCA8, TG, DUOX2, DUOXA2, SLC5A5, and SLC26A4. Variants located in regions with <10× effective coverage were considered low confidence. Such sites were either re-evaluated by repeat sequencing or excluded from downstream analyses unless validated independently by Sanger sequencing. For cases with missing data at specific loci, these positions were treated as unavailable and not used for genotype–phenotype correlation analyses. Only high-confidence variants confirmed by both NGS and, when necessary, Sanger sequencing were included in the final dataset. The “clean reads” (with a length of 90 bp) were derived from targeted sequencing and filtering and subsequently aligned to the human genome reference (hg19) using the Burrows Wheeler Aligner (BWA) MultiVision software package. All SNVs and indels were filtered and estimated via multiple databases, including the 1000 Genomes Project (1KGP), Genome Aggregation Database (gnomAD), Human Gene Mutation Database (HGMD) and Clinvar. We used dbNSFP, which contains seven well-established in silico prediction programs (scale-invariant feature transform (SIFT), Polyphen2, LRT, Mutation Taster, and PhyloP), to predict the effect of missense variants. The pathogenicity of mutations was interpreted based on American College of Medical Genetics and Genomics (ACMG) guidelines (16). All mutations and potential pathogenic variants were validated using conventional Sanger sequencing methods.

Treatment, regular follow-up, and monitoring of confirmed cases were conducted by pediatric endocrinologists from the Newborn Screening Center. Several highly compliant patients received standardized outpatient follow-up and treatment, including assessments of height, weight, head circumference, Gesell development schedules, and L-T4 doses at 1, 2, and 3 years of age. All CH children temporarily stopped L-T4 treatment at the age of 2 years as part of the standardized treatment protocol. Thyroid function was tested after one month. If TSH and FT4 levels were abnormal, the original treatment dosage was resumed. These CH children were diagnosed with PCH and continued to receive regular treatment and follow-up. CH children who stopped L-T4 treatment and maintained normal thyroid function continuously for more than one year were diagnosed with TCH.

Statistical analysis was conducted using SPSS 21.0 software and GraphPad Prism 8 software. We summarized normally distributed quantitative variables using means and standard deviations (means ± SDs). For nonnormally distributed variables, we employed medians with interquartile intervals (Q1-Q3). Count data are expressed as the number of patients (percentages) [N (%)]. The normality of continuous data was assessed using the Shapiro–Wilk test. For normally distributed data with homogeneity of variance, the independent samples t-test or one-way analysis of variance (ANOVA) followed by Bonferroni post hoc correction was used for intergroup comparisons. The Kruskal–Walli’s test was used for nonnormally distributed continuous data. Comparison of count data was performed using the chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered to indicate statistical significance. Graphs were generated using GraphPad Prism 8 software.

Our cohort comprised a total of 117 patients with CH, 91 of whom were identified as carriers of variations in CH-related genes; these patients included 46 males and 45 females. No statistically significant differences were detected between the male and female patients in terms of ethnic distribution, birth age, birth weight, birth height, age at diagnosis, maternal thyroid function, TSH levels at newborn screening (NBS), diagnostic serum TSH levels, diagnostic serum FT4 levels, initial L-T4 doses, thyroid morphology, or clinical outcome (Table 1).

Among the 91 patients, 39 had a normal thyroid, 45 had an enlarged thyroid, and 7 exhibited thyroid dysgenesis; 4 had thyroid agenesis, and 3 had orthotopic hypoplasia. After more than 2 years of treatment and follow-up, 78 cases were classified as PCH, whereas 13 cases were classified as TCH.

The detailed general information, biochemical test results, treatment results, clinical outcomes, and family molecular test results for the 91 patients are provided in Supplementary Table 1. Among them, 67 patients carried DUOX2 gene variations, 9 carried DUOXA2 gene variations, 6 carried TG gene variations, 6 carried TSHR gene variations, 5 carried TPO gene variations, and 2 each carried variations in the GLIS3, LHX3, PAX8, and TBL1X genes. Additionally, 1 patient each carried variations in the CDCA8, IGSF1, IYD, LEPR, POU1F1, SLC26A4, SLC5A5, and TRHR genes (Figure 1). Clearly, DUOX2 gene variations had the highest frequency in this cohort, accounting for 61.5% (67/109) of the total. The next most common variations involved DUOXA2, TG, TSHR, and TPO, which were associated with 8.3% (9/109), 5.5% (6/109), 5.5% (6/109), and 4.6% (5/109), respectively. A total of 47 variant types and 117 variant frequencies were identified in the DUOX2 gene, with c.1588A>T (p.K530X), c.2654G>T (p.R885L), c.3329G>A (p.R1110Q), c.2048G>T (p.R683L), c.3693 + 1G>T, c.2921G>A (p.R974H), and c.4027C>T (p.L1343F) accounting for 12.8%, 10.2%, 10.2%, 6.8%, 5.9%, 5.1%, and 4.2%, respectively. The most common DUOXA2 gene variant was c.738C>G (p.Y246X), accounting for 70.0% (7/10) of the total cases. Notably, digenic variants were found in 11 patients, and oligogenic variants were found in 3 patients. Among gene combinations, DUOX2+TG was the most common, accounting for 27.3% (3/11).

The CH patients were grouped according to thyroid morphology, and we conducted statistical analysis of differences in biochemical test results and clinical outcomes among the groups. We found statistically significant differences (P< 0.001) among the “normal”, “goiter”, and “dysgenesis” groups in terms of TSH levels at NBS, diagnostic serum TSH levels, and diagnostic serum FT4 levels. The “goiter” group and the “dysgenesis” group exhibited higher TSH levels at NBS and diagnostic serum TSH levels, corresponding to lower diagnostic serum FT4 levels. Moreover, the “goiter” group and the “dysgenesis” group required higher initial L-T4 doses than did the “normal” group. However, there was no statistically significant difference in clinical outcomes among the groups (P>0.05) (Table 2, Figure 2).

DUOX2 genotypes were divided into monoallelic variant and biallelic variant groups, and statistical analysis was conducted to assess differences between these two groups in terms of biochemical results, initial L-T4 doses, thyroid morphology, and clinical outcomes. There were no significant differences in TSH levels at NBS, diagnostic serum TSH levels, diagnostic serum FT4 levels, initial L-T4 doses, or clinical outcomes (all P> 0.05). However, there was a significant difference in thyroid morphology between the two groups (P = 0.05). A greater proportion of patients in the “DUOX2 monoallelic variant” group had normal thyroid morphology (50.00%, 10/20), whereas a greater proportion of patients in the “DUOX2 biallelic variant” group had goiter (59.57%, 28/47) (Table 3, Figure 3).

To analyze relationships between genotypes and clinical phenotypes, the 91 CH patients were divided into four groups based on the type of gene variant: those carrying only one monoallelic variant were classified as the “monoallelic variant” group; those with two or more monoallelic variants were in the “more than one monoallelic variant” group; those with only one biallelic variant were in the “biallelic variant” group; and those with one or more monoallelic variants in addition to one biallelic variant were in the “biallelic variant + one or more monoallelic variant” group. The numbers of patients in these four groups were 32, 6, 45, and 8, respectively. There were no significant differences among the four groups in terms of TSH levels at NBS, diagnostic serum TSH levels, diagnostic serum FT4 levels, initial L-T4 doses, or clinical outcomes (P = 0.100, 0.528, 0.570, 0.348, 0.684, respectively). However, there was a significant difference in thyroid morphology among the four groups (P = 0.003). The “monoallelic variant” group had a greater proportion of individuals with “normal” thyroid morphology, accounting for 53.1% (17/32) of the population. With an increase in the number of gene variants, the thyroid morphology gradually shifted toward “goiter” and “dysgenesis” (Table 4, Figure 4).

Among the 91 CH patients, 25 underwent standardized physical and intelligence assessments at the ages of 1, 2, and 3 years. The clinical outcomes included 23 cases of PCH and 2 cases of TCH. The physical development (height, weight, head circumference) and intellectual development (gross motor skills, fine motor skills, adaptive behavior, language, and personal-social behavior) of these patients were within the normal range, showing no significant differences compared to those of same-age children. The patients were divided into three groups based on the types of gene variants they carried. At the same monitoring time points, there were no significant differences in the above indicators among the patients with different gene variant types (all P> 0.05). Moreover, no significant differences in L-T4 doses at the maintenance level at the age of 3 years were detected among the three groups (all P> 0.05). A subset of 25 patients had complete standardized follow-up records at 1, 2, and 3 years; others were excluded due to incomplete data or drop-out (Table 5).