Shiyu Luo

Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients

BACKGROUND Defects in the human dual oxidase 2 (DUOX2) gene are reported to be one of the major causes of congenital hypothyroidism (CH). This study was set to examine the DUOX2 mutation spectrum and prevalence among Chinese CH and subclinical congenital hypothyroidism (SCH) patients and to define the relationships between DUOX2 genotypes and clinical phenotypes. METHODS Peripheral venous blood samples were collected from 192 CH/SCH patients in Guangxi Zhuang Autonomous Region of China. All exons and their exon-intron boundary sequences of the 11 known CH associated genes including DUOX2 were screened by next-generation sequencing (NGS). RESULTS NGS analysis of DUOX2 revealed 18 rare non-polymorphic variants in 57 CH/SCH patients. Sequencing of other CH candidate genes in the 57 patients revealed 2 thyroglobulin (TG) variants. All variants included 11 known mutations, 8 novel variants in DUOX2 and one novel variant in TG, among which three variants p.K530X, p.L1343F and p.R683L are highly recurrent in our patient cohort. 35 (83%) of the 42 patients with one or two DUOX2 pathogenic variants turned out to be SCH or transient congenital hypothyroidism (TCH), whereas 13 (87%) of the 15 patients with three or more DUOX2 pathogenic variants are associated with permanent congenital hypothyroidism (PCH). The accumulation of defects in DUOX2 contribute to the more severe disease regarding thyroid stimulating hormone (TSH) levels, free thyroxine (FT4) levels and initial dose of l-thyroxine (L-T4). CONCLUSION Our study expanded the mutational spectrum of the DUOX2 and TG genes and provided the best estimation of the DUOX2 mutation rate (29%) for CH/SCH patients in Guangxi Zhuang Autonomous Region of China. Most one or two DUOX2 pathogenic variants turned out to be SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly associated with PCH. The coexistence of multiple pathogenic variants may have contributed to the severity of the hypothyroid condition.

Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism.

Objectives Defects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to dyshormonogenesis. The aim of this study was to examine the TPO mutation spectrum and prevalence among patients with CH in the Guangxi Zhuang Autonomous Region of China and to define the relationships between TPO genotypes and clinical phenotypes. Methods Blood samples were collected from 192 patients with CH in the Guangxi Zhuang Autonomous Region, China and genomic DNA was extracted from peripheral blood leucocytes. All exons of the 10 common CH-associated genes including TPO together with their exon-intron boundaries were screened by next-generation sequencing (NGS). The effect of the novel TPO mutation was investigated by ‘in silico’ studies. Results NGS analysis of TPO in 192 patients with CH revealed 3 different variations in 2 individuals (2/192, 1%). Sequencing other CH candidate genes in the patients with TPO variants revealed that patient 1 was homozygous for c.2422delT TPO mutation combined with double heterozygous DUOX2 pathogenic variants (p.R683L/p.L1343F) and patient 2 was triallelic for TPO pathogenic variants (p.R648Q/p.T561M/p.T561M). The present study identified a novel TPO variation c.1682C>T/p.T561M; and four known mutations: c.2422delT/p.C808Afs×24 and c.1943C>T/p.R648Q in TPO, c.2048G>T/p.R683L and c.4027C>T/p.L1343F in DUOX2. Conclusions Our study indicated that the prevalence of TPO mutations was 1% among studied Chinese patients with CH. More than two variations in one or more CH-associated genes can be found in a single patient, and may, in combination, affect the phenotype of the individual. A novel TPO variation c.1682C>T/p.T561M was found, thereby expanding the mutational spectrum of the gene.

Next-generation sequencing analysis of TSHR in 384 Chinese subclinical congenital hypothyroidism (CH) and CH patients

BACKGROUND Defects in the human TSHR gene are reported to be one of the causes of CH due to thyroid dysgenesis, the purpose of this study was to examine the TSHR mutation spectrum and prevalence in congenital hypothyroidism (CH) and subclinical congenital hypothyroidism (SCH) patients in the Guangxi Zhuang Autonomous Region of China and to evaluate the genotype-phenotype correlations. METHODS Blood samples were collected from 384 patients including 240 CH and 144 SCH patients in Guangxi, China. Genomic DNA was extracted from peripheral blood leukocytes. All exons of the 11 known CH associated genes including TSHR together with their exon-intron boundaries were screened by next-generation sequencing (NGS). RESULTS NGS analysis of TSHR revealed nine different variants in ten individuals. Six (4.2%) of 144 patients with SCH were found to harbor monoallelic TSHR variants. Four (1.6%) of 240 patients with CH harbored TSHR variants combined with another monoallelic mutation in either DUOX2 or TG gene. The present study identified five novel variants c.1838A>G (p.Y613C), c.1576G>A (p.A526T), c.2087T>G (p.F696C), c.1631G>A (p.G544E) and c.2051C>A (p.A684D) in TSHR, seven known pathogenic variants c.1349G>A (p.R450H), c.326G>A (p.R109Q), c.2066T>G (p.V689G) and c.2272G>A (p.E758K) in TSHR, IVS3+2T>G in TG, and c.1588A>T (p.K530X) and c.2635G>A (p.E879K) in DUOX2. The previously reported hotspot mutation p.R450H was found in only one SCH patient. CONCLUSION The prevalence of TSHR mutations was 1.6% in CH patients and 4.2% in SCH patients in Guangxi Zhuang Autonomous Region of China. Monoallelic TSHR pathogenic variants were associated with SCH, while TSHR pathogenic variants combined with monoallelic mutations in DUOX2 or TG gene might contribute to CH. Our study expands the TSHR mutation spectrum and provides the best estimation of mutation rate for SCH and CH patients in this Chinese population.

PAX8 pathogenic variants in Chinese patients with congenital hypothyroidism.

BACKGROUND The clinical presentation of patients with congenital hypothyroidism (CH) caused by paired box gene 8 (PAX8) pathogenic variants is variable and PAX8 mutation rates differ significantly among different populations. This study was set to examine the PAX8 mutation spectrum and prevalence among patients with CH in Guangxi Zhuang Autonomous Region, China. METHODS Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes. All exons of the 11 known CH associated genes including PAX8 together with their exon-intron boundaries were screened by next-generation sequencing (NGS). Permanent or transient CH was determined using the results of thyroid function tests after temporary withdrawal of L-thyroxine (L-T4) therapy at approximately 2 years of age. RESULTS Next generation sequencing analysis of PAX8 in 378 CH patients revealed five different mutations in nine individuals (two are siblings). The mutations included two known missense variants, namely c.92G>A (p.R31H) and c.91C>T (p.R31C), and one novel missense variant c.68G>T (p.G23V), as well as two novel nonsense variants c.1090C>T (p. R364X) and c.658C>T (p.R220X). The variant c.92G>A (p.R31H) is highly recurrent in our patient cohort but the clinical phenotypes vary greatly among those carrying this variant. PAX8 pathogenic variants were mainly associated with permanent CH. CONCLUSION The prevalence of PAX8 pathogenic variants was 2.38% among patients with CH in Guangxi. Our study expanded the PAX8 mutation spectrum and provided the best estimation of PAX8 mutation rate among CH patients in Guangxi, China.

Next-generation sequencing analysis of twelve known causative genes in congenital hypothyroidism

BACKGROUND Gene variants have been reported to be associated with congenital hypothyroidism (CH), the purpose of this study was to analyze the mutation spectrum and prevalence of 12 known causative genes (TSHR, PAX8, NKX2.1, NKX2.5, FOXE1, DUOX2, TG, TPO, GLIS3, NIS, SLC26A4 and DEHAL1) in CH in China. METHODS Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes. All exons and their exon-intron boundary sequences of the 12 known CH associated genes in 66 CH patients were screened by next-generation sequencing (NGS). RESULTS NGS analysis of 12 known CH associated genes revealed that 32 patients (32/66, 48.5%) were detected to have at least one potentially functional variant. 21, 9, 1, 1, 1 and 1 patients were found to have potential pathogenic variants in DUOX2, TG, PAX8, SLC26A4, TSHR and TPO genes, respectively. Novel variants included one DUOX2 and one TPO missense variants of unknown significance (VUS). CONCLUSION Our study expands the mutation spectrum of DUOX2 and TPO genes. 48.5% CH patients had at least one potential pathogenic variant. We found relatively high frequency of DUOX2 (31.8%) and TG (13.6%) mutations in our cohort.

Mutation screening of the GLIS3 gene in a cohort of 592 Chinese patients with congenital hypothyroidism

OBJECTIVES Defects in the human GLI-similar 3 (GLIS3) gene are reported to be a rare cause of congenital hypothyroidism (CH) and neonatal diabetes. The aim of this study was to examine the prevalence of GLIS3 mutation among CH patients in the Guangxi Zhuang Autonomous Region of China and to define the relationships between GLIS3 genotypes and clinical phenotypes. METHODS Blood samples were collected from 592 patients with CH in Guangxi Zhuang Autonomous Region, China, and genomic DNA was extracted from peripheral blood leukocytes. All exons of the GLIS3 gene with their exon-intron boundaries were screened by next-generation sequencing (NGS) and CNVplex®. Chromosomal microarray analysis (CMA) was performed to detect the existence of the adjacent gene deletion. RESULTS NGS and CNVplex® analysis of GLIS3 in 592 CH patients revealed two different variations in two individuals (2/592, 0.3%). Patient 1 was the paternal allele of 9p24.3p23 heterozygous deletion including the whole GLIS3 gene, and patient 2 was heterozygous for c.2159G>A (p.R720Q) GLIS3 variant combined with compound heterozygous DUOX2 mutations (p.R683L/p.L1343F). CONCLUSIONS Our study indicated that the prevalence of GLIS3 variations was 0.3% among studied Chinese CH patients. Multiple variations in one or more CH associated genes can be found in one patient. We found a novel GLIS3 variation c.2159G>A (p.R720Q), thereby expanding the variation spectrum of the gene.

Maternal uniparental disomy 14 and mosaic trisomy 14 in a Chinese boy with moderate to severe intellectual disability

BackgroundBoth maternal uniparental disomy 14 (UPD(14)mat) and mosaic trisomy 14 are rare events in live individuals. A combination of the two events in one individual is rarely encountered. Only six live-born cases have so far been reported.Case presentationHere we reported a case of concomitant UPD(14)mat and mosaic trisomy 14 in a 10-year-old Chinese patient. Most clinical features of our patient were consistent with those previous reported for UPD(14)mat cases, which include prenatal and postnatal growth retardation, neonatal hypotonia, feeding difficulty, intellectual disability, truncal obesity, small hands and feet, short stature, and mild facial dysmorphism, but our patient showed more severe intellectual disability and no sign of precocious puberty. SNP array analysis revealed a mixture of chromosome 14 maternal isodisomy with heterodisomy and a low level trisomy mosaicism of whole chromsome 14 in blood and hyperpigmented skin samples, whereas only UPD(14)mat was detected in normal skin sample. Cytogenetic analysis identified one trisomy 14 cell in 100 metaphase of peripheral blood lymphocytes (47,XX, +14[1]/46,XX[99]).ConclusionsTo our knowledge, this is the first case of a patient with UPD(14)mat and mosaic trisomy 14 reported in a Chinese patient. The definitive genetic diagnosis is beneficial for genetic counseling and clinical management of our patient, and for improving our understanding of the genotype-phenotype correlations of concomitant UPD(14)mat and mosaic trisomy 14.

Newborn screening of glucose-6-phosphate dehydrogenase deficiency in Guangxi, China: determination of optimal cutoff value to identify heterozygous female neonates

The aim of this study is to assess the disease incidence and mutation spectrum of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangxi, China, and to determine an optimal cutoff value to identify heterozygous female neonates. A total of 130, 635 neonates were screened from the year of 2013 to 2017. Neonates suspected for G6PD deficiency were further analyzed by quantitatively enzymatic assay and G6PD mutation analysis. The overall incidence of G6PD deficiency was 7.28%. A total of 14 G6PD mutations were identified, and different mutations lead to varying levels of G6PD enzymatic activities. The best cut-off value of G6PD activity in male subjects is 2.2 U/g Hb, same as conventional setting. In female population, however, the cut-off value is found to be 2.8 U/g Hb (sensitivity: 97.5%, specificity: 87.7%, AUC: 0.964) to best discriminate between normal and heterozygotes, and 1.6 U/g Hb (sensitivity: 82.2%, specificity: 85.9%, AUC: 0.871) between heterozygotes and deficient subjects. In conclusion, we have conducted a comprehensive newborn screening of G6PD deficiency in a large cohort of population from Guangxi, China, and first established a reliable cut-off value of G6PD activity to distinguish heterozygous females from either normal or deficient subjects.

The incidence of congenital hypothyroidism (CH) in Guangxi, China and the predictors of permanent and transient CH

Background The incidence of congenital hypothyroidism (CH) differs significantly among different ethnicities and regions, and early differentiation of transient CH is important to avoid unnecessary prolonged treatment with L-T4. Objective To investigate the incidence of CH based on the newborn screening program in Guangxi Zhuang Autonomous Region, China, and to analyze the predictors that might allow for an early differentiation between permanent (P) and transient (T) CH. Design and methods Data from newborn screening program over a seven-year period (January 2009 to January 2016) at Guangxi Maternal and Child Health Hospital are analyzed. Blood samples were collected on filter paper between 3 and 7 days after birth, and TSH level was measured by time-resolved fluorescence assay. Individuals with increased TSH (TSH ≥ 8 IU/L) levels detected by newborn screening were recalled for further evaluation. Serum TSH, FT3 and FT4 were determined by electrochemiluminescence assay using venous blood samples. Diagnosis of CH is based on elevated TSH levels (>10 IU/L) and decreased FT4 levels (<12 pmol/L). Patients with elevated TSH levels and normal FT4 levels were diagnosed as hyperthyrotropinemia. Permanent or transient CH was determined by using the results of thyroid function tests after temporary withdrawal of L-T4 therapy at approximately 2–3 years of age. Results Among 1,238,340 infants in the newborn screening program, 14,443 individuals were recalled for reevaluation (re-call rate 1.18%), 911 and 731 individuals were subsequently determined to have hyperthyrotropinemia and CH respectively; thus, a prevalence of 1:1359 and 1:1694 for hyperthyrotropinemia and CH. Of the 731 patients with CH, 161 patients were diagnosed with permanent CH (PCH), and 159 patients were diagnosed with transient CH (TCH), the other 411 patients are too young to determine their subtypes. Patients with PCH required an increasing dose of L-T4 during the first few years, whereas patients with TCH required a decreased dose of L-T4. The TSH levels at diagnosis and the dose of L-T4 used were significantly higher in PCH cases than in transient cases. The FT4 levels at diagnosis were significantly lower in PCH cases than in TCH cases. The TSH levels at diagnosis, FT4 levels at diagnosis and L-T4 doses at 90 days were evaluated as predictors for differentiating PCH and TCH, and their accuracy at their respective optimal cutoffs were determined to be 60.6%, 66.7% and 93.9%, respectively. Conclusions The CH incidence in Guangxi Zhuang Autonomous Region is slightly higher (1:1694) compared to the worldwide levels (1/2000–1/4000). The PCH and TCH ratio is close to 1; thus, the estimated PCH incidence is 1/3388, which is similar to reported worldwide average incidence (1/3000). The L-T4 dose required at 90 days (>30 μg/day) has the highest predictive value for PCH. Earlier differentiation of PCH and TCH helps to determine appropriate treatment course.

Novel and reported APC germline mutations in Chinese patients with familial adenomatous polyposis.

OBJECTIVE Familial adenomatous polyposis (FAP) is mainly caused by germline mutations in the adenomatous polyposis coli (APC) gene. This study aimed to detect pathogenic variants in five Chinese FAP families and review all previously reported pathogenic variants of APC gene in Chinese population. METHODS Five non-consanguineous FAP families and 100 unrelated ethnicity-matched controls were included in the study. Sanger sequencing was performed to screen for APC coding and splicing variants. Chinese and English literature on APC germline mutations were reviewed to compile the mutation spectrum of APC gene in Chinese FAP patients. RESULTS One pathogenic variant was detected in each family for the five pedigrees we tested. Three variants (c.3183_3187delACAAA, c.2626C>T and c.1312+1G>A) were previously reported as pathogenic. The other two variants were novel: c.794_795insG/p.Val266SerfsTer11 and c.2142_2143insG/p.His715AlafsTer19. They are absent from public databases (1000 Genomes, dbSNP, ESP and ExAC) and 100 normal controls, and are classified as pathogenic based on the new ACMG/AMP variant classification guidelines. Literature review and current study revealed a total of 82 different pathogenic variants from 127 Chinese FAP families. Among these families, 83 families had frameshift variants (65.35%), 26 with nonsense variants (20.47%), six with splice site variants (4.72%), three with missense variants (2.36%) and nine with large deletion or duplication variants (7.09%). Apart from the two previously reported mutation hotspots c.3927_3931delAAAGA (20.47%) and c.3183_3187delACAAA (7.09%), c.847C>T/p.Arg283Ter variant occurred with a frequency of 3.15% (4 out of 127) in Chinese FAP patients. CONCLUSIONS We reported two novel pathogenic variants. The comprehensive compilation of variants and comparison revealed largely similar mutation spectrum between Chinese and Western patient populations. Some unique features noticed in Chinese patient population may help to better understand the pathogenesis of FAP.