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Mutation screening of the GLIS3 gene in a cohort of 592 Chinese patients with congenital hypothyroidism
Clinica Chimica Acta 476 (2018) 38–43
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Mutation screening of the GLIS3 gene in a cohort of 592 Chinese patients with congenital hypothyroidism
T
Chunyun Fua,b,c,d,1, Shiyu Luoc,d,1, Xigui Longe,1, Yingfeng Lia,d, Shangyang Shea,d, Xuehua Hua,d, Meizhen Mob, Zhanghong Wangb, Yuhua Chenb, Chun Heb, Jiasun Suc, Yue Zhangc, Fei Linc, ⁎ ⁎ Bobo Xiec, Qifei Lic,f, , Shaoke Chenc,f, a
Medical Science Laboratory, Children's Hospital, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China Department of Pathology, Children's Hospital, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China c Department of Genetic Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China d Birth Defects Prevention and Control Institute of Guangxi Zhuang Autonomous Region, Nanning 530003, China e National Laboratory of medical Genetics, Central South University, Changsha, 410000, China f Guangxi Huayin Medical Laboratory Center, Nanning 530012, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Congenital hypothyroidism GLIS3 Gene mutations China Next-generation sequencing
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.
1. Introduction Defects in the human GLI-similar 3 (GLIS3) gene (NM_001042413) are reported to be one of the rare cause of congenital hypothyroidism (CH) and neonatal diabetes [1–4]. GLIS3, a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2type zinc finger domains, maps to chromosome 9p24.3-p23. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. It is specifically involved in the development of pancreatic β-cells, the thyroid, eye, liver, and kidney although tissue expression occurs to a lesser extent in the heart, skeletal muscle, stomach, brain, adrenal gland, and bone [2,5]. GLIS3 gene was also reported to be a candidate imprinted gene, which is
⁎
1
paternally expressed in human placenta [6]. Patients with GLIS3 mutations presented with a wider phenotype consisting mainly of neonatal diabetes and CH, in addition to multiple features involving different organs [7]. Up to now, 19 CH patients caused by GLIS3 mutations were reported with an autosomal recessive inheritance pattern, and partial gene deletions as the most common type of GLIS3 mutations [7–11]. Given the rarity of this condition, the genotype-phenotype relationships has not yet been fully established, and little is known about its mutational spectrum and prevalence among Chinese CH patients. Here we performed the GLIS3 gene screening in a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China.
Corresponding authors at: Department of Genetic Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China. E-mail addresses: [email protected] (Q. Li), [email protected] (S. Chen). These authors contributed equally.
https://doi.org/10.1016/j.cca.2017.11.011 Received 14 September 2017; Received in revised form 23 October 2017; Accepted 13 November 2017 Available online 13 November 2017 0009-8981/ © 2017 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 476 (2018) 38–43
C. Fu et al.
Fig. 1. CNVplex® analysis of GLIS3 gene. A) patient 1 was detected to be heterozygous for Exons 1-11 del/- GLIS3 mutation; B) normal control.
2. Materials and methods
In addition, a cohort of 600 ethnicity-matched healthy participants was used to assess the variant frequencies in normal control. All control participants had normal FT4 and TSH levels.
2.1. Patients A total of 592 patients were enrolled in this study, who were identified through newborn screening among 1,143,000 newborns in the Guangxi Zhuang Autonomous Region of China from June 2009 to June 2016. The methods of newborn screening of CH have been described in detail previously [12,13]. This study was approved by the local Medical Ethics Committee of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. Written informed consent was obtained from the parents of the patients.
2.3. Sanger sequencing Sanger sequencing was used to validate the variants identified from next-generation sequencing. 3. Results 3.1. Next generation sequencing and CNVplex® analysis of GLIS3 and other CH associated genes
2.2. Mutation detection and interpretation In this study, we identified two different GLIS3 variants in two individuals. Sequencing of other CH candidate genes in the 2 patients with the GLIS3 variations showed that patient 1 was heterozygous for Exons 1-11 del/- GLIS3 mutation (Fig. 1), and patient 2 was heterozygous for c.2159G > A (p.R720Q) GLIS3 variation combined with compound heterozygous DUOX2 mutations [c.2048G > T (p.R683L)/ c.4027G > T (p.L1343F)]. All missense variants were confirmed by Sanger sequencing (Fig. 2). The present study identified a novel GLIS3 variation c.2159G > A (p.R720Q) that was not detected in our normal control population and classified as variant of uncertain significance (VUS) according to the ACMG/AMP guideline (Table 1). CMA analysis was performed using the Illumina HumanSNPcyto-12
Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. All exons of the GLIS3 gene with their exonintron boundaries were screened by NGS and CNVplex®. CNVplex® is based on the MLPA method, it can be used for the detection of chromosomal alterations [14]. When the whole GLIS3 gene deletions were found, chromosomal microarray analysis (CMA) was later performed in order to detect the presence of the adjacent gene deletion. The methods and workflows of NGS [15], CMA [16] and CNVplex® [14] have been described in detail previously. 39
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Fig. 2. DNA Sanger sequencing for validation of the variations detected in patient 2 by next generation sequencing.
Table 1 Classification and evidence of the novel GLIS3 variant: c.2159G > A (p.R720Q). Variant
c.2159G > A (p.R720Q)
Gene
GLIS3
Classification
VUS
PS4
PM1
PM2
PP3
Frequency in 600 normal controls
Located in functional domain
Frequency in East Asian Population (and in globe)
In-silico prediction
0
NA
0.001513 (0.0001405)
NA
Classification and evidence system according to ACMG/AMP variants interpretation guidelines. PS4 = The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. PM1 = Located in a mutational hot spot and/or critical and well-established functional domain. PM2 = Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC. PP3 = Multiple lines of computational evidence support a deleterious effect on the gene or gene product. VUS = Variants of Uncertain Significance; NA = Data not available.
hypertelorism, flat nasal bridge, low posterior hairline, camptodactyly, syndactyly and polydactyly (Fig. 4). Family studies by CMA analysis revealed that the deletion was de novo and come from the paternal allele. Patient 2 was diagnosed as CH by newborn screening. Ultrasound examination showed an increased size thyroid gland (right lobe: 3.4 × 1.5 × 1.4 cm; left lobe: 3.4 × 1.4 × 1.3 cm). L-T4 replacement therapy was started immediately at an initial daily dose of 12 μg/kg. He was diagnosed as PCH due to a high TSH level (40.5mIU/L) after temporary withdrawal of L-T4 therapy for 4 weeks at the age of 2.9 years. The L-T4 replacement therapy was resumed. The patient is now 3.5 years of age and receives a daily dose of 7 μg/kg L-T4. His physical and intellectual development is appropriate to his age, no other abnormal phenotype was found. Family studies showed that his father carried a heterozygous DUOX2 mutation p. R683L but with no thyroid phenotype, and his mother harboured the heterozygous DUOX2 mutation p.L1343F without abnormal thyroid phenotypes.
v2.1 BeadChip array (Illumina Inc. San Diego, CA, USA). This array contains about 300,000 single nucleotide polymorphisms (SNPs) probes covering the human genome (NCBI build GRCh37/hg19). CMA testing of patient 1's genomic DNA (Fig. 3) detected a paternal allele of 13 Mb deletion in the 9p24.3p23 region, ranging from position 46,587 to 13,267,800. The deleted 9p24.3p23 region contains 38 known genes, among the affected genes the following have been associated with genetic disorders: DOCK8, KANK1, SMARCA2, VLDLR, KCNV2, CLIS3, SLC1A1, JAK2, GLDC, TYRP1 and MPD2. Both father's and mother's CMA results were normal. 3.2. Clinical features and laboratory test results of the patients Patient 1: This patient was diagnosed as CH at the age of 5 months in our pediatric clinic, and the thyroid anatomy was normal on ultrasonography. L-T4 treatment was started immediately after diagnosis at an initial daily dose of 12 μg/kg. The patients was diagnosed as permanent congenital hypothyroidism (PCH) since she still had high TSH levels that did not reduce to normal limits with L-T4 therapy. The patient is now 9 years and 10 months of age and receives a daily dose of 1.8 μg/kg L-T4. Her birth weight was 8000 g and birth length was 140.5 cm. The patient shows delayed motor development, learning difficulties and diabetes with prominent forehead, low-set ears,
4. Discussion In the present study, we conducted the largest GLIS3 gene mutation screening in 592 CH patients from Guangxi Zhuang Autonomous Region by NGS and CNVplex®. The result of our study identified two 40
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Fig. 3. CMA testing result of Patient 1. A 13 Mb deletion in the 9p24.3p23 region was detected.
variation and compound heterozygous DUOX2 mutations. This novel GLIS3 variation in patient 2 was evaluated as VUS according to the ACMG/AMP guideline although further functional studies may provide more evidence to elucidate its significance. It was proposed that the clinical phenotype in this patient was caused by DUOX2 mutations because he had only CH symptom. Our study also demonstrated that multiple variations in one or more CH associated gene can be found in one patient. Mutations in the GLIS3 gene are a rare cause of neonatal diabetes and congenital hypothyroidism. Additional features, as previously described, include facial dysmorphism (17/19), developmental delay (14/ 19), liver diseases (14/19), kidney diseases (13/19), congenital glaucoma (9/19), pancreatic exocrine insufficiency (6/19), sensorineural deafness (5/19), osteopenia (2/19), pancreatic cysts (2/19), recurrent infections (2/19), microphallus (1/19), patent ductus arteriosus (1/19) and scrotal hypospadias (1/19). In agreement with previous reports of GLIS3 mutations, patient 1 had diabetes, congenital hypothyroidism, facial dysmorphism and developmental delay but she also presented with some phenotypes that have not been described, such as camptodactyly, syndactyly and polydactyly. Our study thus expanded the clinical phenotype associated with GLIS3 mutation.
different GLIS3 variations in two CH patients, and revealed a GLIS3 variation rate of 0.3%, once again demonstrating GLIS3 gene mutation to be a rare cause of CH. Up to now, 19 CH patients caused by GLIS3 mutations have been reported, all cases of CH associated with alterations in the GLIS3 gene are caused by either homozygous or compound heterozygous mutations with an autosomal recessive inheritance pattern [7–9]. In addition, human GLIS3 gene was identified to be a candidate imprinted gene with monoallelic paternally expression in human placenta, but no study was conducted to determine whether it was also imprinted in the human thyroid and other organizations [6]. In this study, patient 1 has a typical GLIS3 phenotype. CNVplex® analysis found patient 1 carried a heterozygous deletion of GLIS3. Familial CMA analysis demonstrated that the heterozygous deletion of GLIS3 was paternal allelic. If GLIS3 gene was also imprinted in the human thyroid and other organizations, since the existing monoallelic maternal copy didn't express, the patient would loss GLIS3 gene function and had disease phenotype. If not, we cannot exclude the possibility that the second allele of GLIS3 also carried a mutation, which would not have been identified if located in an intronic or regulatory sequence. Patient 2 only presented with CH symptom. NGS and CNVplex® testing found that the patient carried both a heterozygous GLIS3 41
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Fig. 4. Illustration of Patient 1's phenotypes, including prominent forehead, hypertelorism, nasal bridge, low posterior hairline, low-set ears, camptodactyly, syndactyly and polydactyly.
Strengths and limitations of this study
Most of GLIS3 mutations are the deletion of GLIS3 gene [8]. We also need to consider the possibility of adjacent gene deletion when the whole GLIS3 gene was deleted. Similarly, the effects of other genes on the clinical phenotype should be considered when establishing the relationship between genotype and phenotype in GLIS3. In this study, the genotype-phenotype correlation according to the type of mutation could not be determined because only two patients were identified with GLIS3 variations and both of them had multiple mutations referred to two or more genes. In conclusion, we conducted one of the largest GLIS3 mutation screening in a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China. We identified the prevalence of GLIS3 variations (0.3% among our CH patients). A novel GLIS3 variation was found. Our study expanded both GLIS3 variation spectrum and clinical phenotype associated with GLIS3 mutation, and indicated that the GLIS3 mutation rate is very low in the Guangxi Zhuang Autonomous Region, China.
▪ We conducted one of the largest GLIS3 mutation screening for a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China. ▪ We identified the prevalence of GLIS3 variations (0.3% among CH patients). ▪ Multiple variations in one or more CH associated genes can be found in one patient. ▪ We found a novel GLIS3 variation, thereby expanding the variation spectrum of the gene. ▪ The relationships between GLIS3 genotypes and clinical phenotypes could not be clearly determined because only two patients were identified to be mutation-positive subjects and both of them had multiple variations in different genes. References
Competing interests
[1] X. Wen, Y. Yang, Emerging roles of GLIS3 in neonatal diabetes, type 1 and type 2 diabetes, J. Mol. Endocrinol. 58 (2017) R73–R85. [2] K. Lichti-Kaiser, G. ZeRuth, H.S. Kang, S. Vasanth, A.M. Jetten, Gli-similar proteins: their mechanisms of action, physiological functions, and roles in disease, Vitam. Horm. 88 (2012) 141–171. [3] Y. Yang, B.H. Chang, S.L. Samson, M.V. Li, L. Chan, The Kruppel-like zinc finger protein Glis3 directly and indirectly activates insulin gene transcription, Nucleic Acids Res. 37 (2009) 2529–2538. [4] F. Barbetti, Diagnosis of neonatal and infancy-onset diabetes, Endocr. Dev. 11 (2007) 83–93. [5] C.K. Chou, C.J. Tang, H.L. Chou, et al., The potential role of Kruppel-like zinc-finger protein Glis3 in genetic diseases and cancers, Arch. Immunol. Ther. Exp. (2017), http://dx.doi.org/10.1007/s00005-017-0470-x. [6] S. Barbaux, G. Gascoin-Lachambre, C. Buffat, et al., A genome-wide approach reveals novel imprinted genes expressed in the human placenta, Epigenetics 7 (2012) 1079–1090. [7] K.A. Alghamdi, A.B. Alsaedi, A. Aljasser, A. Altawil, N.M. Kamal, Extended clinical features associated with novel Glis3 mutation: a case report, BMC Endocr. Disord. 17 (2017) 14. [8] P. Dimitri, A.M. Habeb, F. Gurbuz, et al., Expanding the clinical spectrum
No potential conflicts of interest relevant to this original article were reported by authors. Funding This study was supported by the Guangxi Natural Science Foundation Program (2016GXNSFBA380192 and 2012GXNSFAA053174), National Natural Science Foundation of China (81260126) and Key Projects of Guangxi Health Department (2012025). Data sharing statement We will coordinate data sharing and make available both the raw data and analyzed data upon request. 42
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[9] [10]
[11]
[12]
[13] C. Fu, S. Zhang, J. Su, et al., Mutation screening of DUOX2 in Chinese patients with congenital hypothyroidism, J. Endocrinol. Investig. 38 (2015) 1219–1224. [14] X. Zhang, Y. Xu, D. Liu, et al., A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease, BMC Genomics 16 (2015) 364. [15] C. Fu, S. Luo, S. Zhang, et al., Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients, Clin. Chim. Acta 458 (2016) 30–34. [16] R. Chen, C. Li, B. Xie, et al., Clinical and molecular evaluations of siblings with "pure" 11q23.3-qter trisomy or reciprocal monosomy due to a familial translocation t (10;11) (q26;q23.3), Mol. Cytogenet. 7 (2014) 101.
associated with GLIS3 mutations, J. Clin. Endocrinol. Metab. 100 (2015) E1362–1369. P. Dimitri, J.T. Warner, J.A. Minton, et al., Novel GLIS3 mutations demonstrate an extended multisystem phenotype, Eur. J. Endocrinol. 164 (2011) 437–443. J.Y. Beak, H.S. Kang, Y.S. Kim, A.M. Jetten, Functional analysis of the zinc finger and activation domains of Glis3 and mutant Glis3(NDH1), Nucleic Acids Res. 36 (2008) 1690–1702. V. Senee, C. Chelala, S. Duchatelet, et al., Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism, Nat. Genet. 38 (2006) 682–687. C. Fu, B. Xie, S. Zhang, et al., Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism, BMJ Open 6 (2016) e010719.
43
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Mutation screening of the GLIS3 gene in a cohort of 592 Chinese patients with congenital hypothyroidism
T
Chunyun Fua,b,c,d,1, Shiyu Luoc,d,1, Xigui Longe,1, Yingfeng Lia,d, Shangyang Shea,d, Xuehua Hua,d, Meizhen Mob, Zhanghong Wangb, Yuhua Chenb, Chun Heb, Jiasun Suc, Yue Zhangc, Fei Linc, ⁎ ⁎ Bobo Xiec, Qifei Lic,f, , Shaoke Chenc,f, a
Medical Science Laboratory, Children's Hospital, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China Department of Pathology, Children's Hospital, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China c Department of Genetic Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China d Birth Defects Prevention and Control Institute of Guangxi Zhuang Autonomous Region, Nanning 530003, China e National Laboratory of medical Genetics, Central South University, Changsha, 410000, China f Guangxi Huayin Medical Laboratory Center, Nanning 530012, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Congenital hypothyroidism GLIS3 Gene mutations China Next-generation sequencing
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.
1. Introduction Defects in the human GLI-similar 3 (GLIS3) gene (NM_001042413) are reported to be one of the rare cause of congenital hypothyroidism (CH) and neonatal diabetes [1–4]. GLIS3, a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2type zinc finger domains, maps to chromosome 9p24.3-p23. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. It is specifically involved in the development of pancreatic β-cells, the thyroid, eye, liver, and kidney although tissue expression occurs to a lesser extent in the heart, skeletal muscle, stomach, brain, adrenal gland, and bone [2,5]. GLIS3 gene was also reported to be a candidate imprinted gene, which is
⁎
1
paternally expressed in human placenta [6]. Patients with GLIS3 mutations presented with a wider phenotype consisting mainly of neonatal diabetes and CH, in addition to multiple features involving different organs [7]. Up to now, 19 CH patients caused by GLIS3 mutations were reported with an autosomal recessive inheritance pattern, and partial gene deletions as the most common type of GLIS3 mutations [7–11]. Given the rarity of this condition, the genotype-phenotype relationships has not yet been fully established, and little is known about its mutational spectrum and prevalence among Chinese CH patients. Here we performed the GLIS3 gene screening in a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China.
Corresponding authors at: Department of Genetic Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China. E-mail addresses: [email protected] (Q. Li), [email protected] (S. Chen). These authors contributed equally.
https://doi.org/10.1016/j.cca.2017.11.011 Received 14 September 2017; Received in revised form 23 October 2017; Accepted 13 November 2017 Available online 13 November 2017 0009-8981/ © 2017 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 476 (2018) 38–43
C. Fu et al.
Fig. 1. CNVplex® analysis of GLIS3 gene. A) patient 1 was detected to be heterozygous for Exons 1-11 del/- GLIS3 mutation; B) normal control.
2. Materials and methods
In addition, a cohort of 600 ethnicity-matched healthy participants was used to assess the variant frequencies in normal control. All control participants had normal FT4 and TSH levels.
2.1. Patients A total of 592 patients were enrolled in this study, who were identified through newborn screening among 1,143,000 newborns in the Guangxi Zhuang Autonomous Region of China from June 2009 to June 2016. The methods of newborn screening of CH have been described in detail previously [12,13]. This study was approved by the local Medical Ethics Committee of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. Written informed consent was obtained from the parents of the patients.
2.3. Sanger sequencing Sanger sequencing was used to validate the variants identified from next-generation sequencing. 3. Results 3.1. Next generation sequencing and CNVplex® analysis of GLIS3 and other CH associated genes
2.2. Mutation detection and interpretation In this study, we identified two different GLIS3 variants in two individuals. Sequencing of other CH candidate genes in the 2 patients with the GLIS3 variations showed that patient 1 was heterozygous for Exons 1-11 del/- GLIS3 mutation (Fig. 1), and patient 2 was heterozygous for c.2159G > A (p.R720Q) GLIS3 variation combined with compound heterozygous DUOX2 mutations [c.2048G > T (p.R683L)/ c.4027G > T (p.L1343F)]. All missense variants were confirmed by Sanger sequencing (Fig. 2). The present study identified a novel GLIS3 variation c.2159G > A (p.R720Q) that was not detected in our normal control population and classified as variant of uncertain significance (VUS) according to the ACMG/AMP guideline (Table 1). CMA analysis was performed using the Illumina HumanSNPcyto-12
Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. All exons of the GLIS3 gene with their exonintron boundaries were screened by NGS and CNVplex®. CNVplex® is based on the MLPA method, it can be used for the detection of chromosomal alterations [14]. When the whole GLIS3 gene deletions were found, chromosomal microarray analysis (CMA) was later performed in order to detect the presence of the adjacent gene deletion. The methods and workflows of NGS [15], CMA [16] and CNVplex® [14] have been described in detail previously. 39
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Fig. 2. DNA Sanger sequencing for validation of the variations detected in patient 2 by next generation sequencing.
Table 1 Classification and evidence of the novel GLIS3 variant: c.2159G > A (p.R720Q). Variant
c.2159G > A (p.R720Q)
Gene
GLIS3
Classification
VUS
PS4
PM1
PM2
PP3
Frequency in 600 normal controls
Located in functional domain
Frequency in East Asian Population (and in globe)
In-silico prediction
0
NA
0.001513 (0.0001405)
NA
Classification and evidence system according to ACMG/AMP variants interpretation guidelines. PS4 = The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. PM1 = Located in a mutational hot spot and/or critical and well-established functional domain. PM2 = Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC. PP3 = Multiple lines of computational evidence support a deleterious effect on the gene or gene product. VUS = Variants of Uncertain Significance; NA = Data not available.
hypertelorism, flat nasal bridge, low posterior hairline, camptodactyly, syndactyly and polydactyly (Fig. 4). Family studies by CMA analysis revealed that the deletion was de novo and come from the paternal allele. Patient 2 was diagnosed as CH by newborn screening. Ultrasound examination showed an increased size thyroid gland (right lobe: 3.4 × 1.5 × 1.4 cm; left lobe: 3.4 × 1.4 × 1.3 cm). L-T4 replacement therapy was started immediately at an initial daily dose of 12 μg/kg. He was diagnosed as PCH due to a high TSH level (40.5mIU/L) after temporary withdrawal of L-T4 therapy for 4 weeks at the age of 2.9 years. The L-T4 replacement therapy was resumed. The patient is now 3.5 years of age and receives a daily dose of 7 μg/kg L-T4. His physical and intellectual development is appropriate to his age, no other abnormal phenotype was found. Family studies showed that his father carried a heterozygous DUOX2 mutation p. R683L but with no thyroid phenotype, and his mother harboured the heterozygous DUOX2 mutation p.L1343F without abnormal thyroid phenotypes.
v2.1 BeadChip array (Illumina Inc. San Diego, CA, USA). This array contains about 300,000 single nucleotide polymorphisms (SNPs) probes covering the human genome (NCBI build GRCh37/hg19). CMA testing of patient 1's genomic DNA (Fig. 3) detected a paternal allele of 13 Mb deletion in the 9p24.3p23 region, ranging from position 46,587 to 13,267,800. The deleted 9p24.3p23 region contains 38 known genes, among the affected genes the following have been associated with genetic disorders: DOCK8, KANK1, SMARCA2, VLDLR, KCNV2, CLIS3, SLC1A1, JAK2, GLDC, TYRP1 and MPD2. Both father's and mother's CMA results were normal. 3.2. Clinical features and laboratory test results of the patients Patient 1: This patient was diagnosed as CH at the age of 5 months in our pediatric clinic, and the thyroid anatomy was normal on ultrasonography. L-T4 treatment was started immediately after diagnosis at an initial daily dose of 12 μg/kg. The patients was diagnosed as permanent congenital hypothyroidism (PCH) since she still had high TSH levels that did not reduce to normal limits with L-T4 therapy. The patient is now 9 years and 10 months of age and receives a daily dose of 1.8 μg/kg L-T4. Her birth weight was 8000 g and birth length was 140.5 cm. The patient shows delayed motor development, learning difficulties and diabetes with prominent forehead, low-set ears,
4. Discussion In the present study, we conducted the largest GLIS3 gene mutation screening in 592 CH patients from Guangxi Zhuang Autonomous Region by NGS and CNVplex®. The result of our study identified two 40
Clinica Chimica Acta 476 (2018) 38–43
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Fig. 3. CMA testing result of Patient 1. A 13 Mb deletion in the 9p24.3p23 region was detected.
variation and compound heterozygous DUOX2 mutations. This novel GLIS3 variation in patient 2 was evaluated as VUS according to the ACMG/AMP guideline although further functional studies may provide more evidence to elucidate its significance. It was proposed that the clinical phenotype in this patient was caused by DUOX2 mutations because he had only CH symptom. Our study also demonstrated that multiple variations in one or more CH associated gene can be found in one patient. Mutations in the GLIS3 gene are a rare cause of neonatal diabetes and congenital hypothyroidism. Additional features, as previously described, include facial dysmorphism (17/19), developmental delay (14/ 19), liver diseases (14/19), kidney diseases (13/19), congenital glaucoma (9/19), pancreatic exocrine insufficiency (6/19), sensorineural deafness (5/19), osteopenia (2/19), pancreatic cysts (2/19), recurrent infections (2/19), microphallus (1/19), patent ductus arteriosus (1/19) and scrotal hypospadias (1/19). In agreement with previous reports of GLIS3 mutations, patient 1 had diabetes, congenital hypothyroidism, facial dysmorphism and developmental delay but she also presented with some phenotypes that have not been described, such as camptodactyly, syndactyly and polydactyly. Our study thus expanded the clinical phenotype associated with GLIS3 mutation.
different GLIS3 variations in two CH patients, and revealed a GLIS3 variation rate of 0.3%, once again demonstrating GLIS3 gene mutation to be a rare cause of CH. Up to now, 19 CH patients caused by GLIS3 mutations have been reported, all cases of CH associated with alterations in the GLIS3 gene are caused by either homozygous or compound heterozygous mutations with an autosomal recessive inheritance pattern [7–9]. In addition, human GLIS3 gene was identified to be a candidate imprinted gene with monoallelic paternally expression in human placenta, but no study was conducted to determine whether it was also imprinted in the human thyroid and other organizations [6]. In this study, patient 1 has a typical GLIS3 phenotype. CNVplex® analysis found patient 1 carried a heterozygous deletion of GLIS3. Familial CMA analysis demonstrated that the heterozygous deletion of GLIS3 was paternal allelic. If GLIS3 gene was also imprinted in the human thyroid and other organizations, since the existing monoallelic maternal copy didn't express, the patient would loss GLIS3 gene function and had disease phenotype. If not, we cannot exclude the possibility that the second allele of GLIS3 also carried a mutation, which would not have been identified if located in an intronic or regulatory sequence. Patient 2 only presented with CH symptom. NGS and CNVplex® testing found that the patient carried both a heterozygous GLIS3 41
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Fig. 4. Illustration of Patient 1's phenotypes, including prominent forehead, hypertelorism, nasal bridge, low posterior hairline, low-set ears, camptodactyly, syndactyly and polydactyly.
Strengths and limitations of this study
Most of GLIS3 mutations are the deletion of GLIS3 gene [8]. We also need to consider the possibility of adjacent gene deletion when the whole GLIS3 gene was deleted. Similarly, the effects of other genes on the clinical phenotype should be considered when establishing the relationship between genotype and phenotype in GLIS3. In this study, the genotype-phenotype correlation according to the type of mutation could not be determined because only two patients were identified with GLIS3 variations and both of them had multiple mutations referred to two or more genes. In conclusion, we conducted one of the largest GLIS3 mutation screening in a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China. We identified the prevalence of GLIS3 variations (0.3% among our CH patients). A novel GLIS3 variation was found. Our study expanded both GLIS3 variation spectrum and clinical phenotype associated with GLIS3 mutation, and indicated that the GLIS3 mutation rate is very low in the Guangxi Zhuang Autonomous Region, China.
▪ We conducted one of the largest GLIS3 mutation screening for a cohort of 592 patients with CH in Guangxi Zhuang Autonomous Region, China. ▪ We identified the prevalence of GLIS3 variations (0.3% among CH patients). ▪ Multiple variations in one or more CH associated genes can be found in one patient. ▪ We found a novel GLIS3 variation, thereby expanding the variation spectrum of the gene. ▪ The relationships between GLIS3 genotypes and clinical phenotypes could not be clearly determined because only two patients were identified to be mutation-positive subjects and both of them had multiple variations in different genes. References
Competing interests
[1] X. Wen, Y. Yang, Emerging roles of GLIS3 in neonatal diabetes, type 1 and type 2 diabetes, J. Mol. Endocrinol. 58 (2017) R73–R85. [2] K. Lichti-Kaiser, G. ZeRuth, H.S. Kang, S. Vasanth, A.M. Jetten, Gli-similar proteins: their mechanisms of action, physiological functions, and roles in disease, Vitam. Horm. 88 (2012) 141–171. [3] Y. Yang, B.H. Chang, S.L. Samson, M.V. Li, L. Chan, The Kruppel-like zinc finger protein Glis3 directly and indirectly activates insulin gene transcription, Nucleic Acids Res. 37 (2009) 2529–2538. [4] F. Barbetti, Diagnosis of neonatal and infancy-onset diabetes, Endocr. Dev. 11 (2007) 83–93. [5] C.K. Chou, C.J. Tang, H.L. Chou, et al., The potential role of Kruppel-like zinc-finger protein Glis3 in genetic diseases and cancers, Arch. Immunol. Ther. Exp. (2017), http://dx.doi.org/10.1007/s00005-017-0470-x. [6] S. Barbaux, G. Gascoin-Lachambre, C. Buffat, et al., A genome-wide approach reveals novel imprinted genes expressed in the human placenta, Epigenetics 7 (2012) 1079–1090. [7] K.A. Alghamdi, A.B. Alsaedi, A. Aljasser, A. Altawil, N.M. Kamal, Extended clinical features associated with novel Glis3 mutation: a case report, BMC Endocr. Disord. 17 (2017) 14. [8] P. Dimitri, A.M. Habeb, F. Gurbuz, et al., Expanding the clinical spectrum
No potential conflicts of interest relevant to this original article were reported by authors. Funding This study was supported by the Guangxi Natural Science Foundation Program (2016GXNSFBA380192 and 2012GXNSFAA053174), National Natural Science Foundation of China (81260126) and Key Projects of Guangxi Health Department (2012025). Data sharing statement We will coordinate data sharing and make available both the raw data and analyzed data upon request. 42
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[9] [10]
[11]
[12]
[13] C. Fu, S. Zhang, J. Su, et al., Mutation screening of DUOX2 in Chinese patients with congenital hypothyroidism, J. Endocrinol. Investig. 38 (2015) 1219–1224. [14] X. Zhang, Y. Xu, D. Liu, et al., A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease, BMC Genomics 16 (2015) 364. [15] C. Fu, S. Luo, S. Zhang, et al., Next-generation sequencing analysis of DUOX2 in 192 Chinese subclinical congenital hypothyroidism (SCH) and CH patients, Clin. Chim. Acta 458 (2016) 30–34. [16] R. Chen, C. Li, B. Xie, et al., Clinical and molecular evaluations of siblings with "pure" 11q23.3-qter trisomy or reciprocal monosomy due to a familial translocation t (10;11) (q26;q23.3), Mol. Cytogenet. 7 (2014) 101.
associated with GLIS3 mutations, J. Clin. Endocrinol. Metab. 100 (2015) E1362–1369. P. Dimitri, J.T. Warner, J.A. Minton, et al., Novel GLIS3 mutations demonstrate an extended multisystem phenotype, Eur. J. Endocrinol. 164 (2011) 437–443. J.Y. Beak, H.S. Kang, Y.S. Kim, A.M. Jetten, Functional analysis of the zinc finger and activation domains of Glis3 and mutant Glis3(NDH1), Nucleic Acids Res. 36 (2008) 1690–1702. V. Senee, C. Chelala, S. Duchatelet, et al., Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism, Nat. Genet. 38 (2006) 682–687. C. Fu, B. Xie, S. Zhang, et al., Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism, BMJ Open 6 (2016) e010719.
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