Research Article
Annals Thyroid Res. 2016; 2(2): 69-78.
Biallelic Stop Codon Mutations (p.F353Pfs*36/p.Y425X) in DUOX2 Gene Associated with Transient Congenital Hypothyroidism: Report of a Family and Literature Review
Fiorella S Belforte¹, María Cecilia Olcese¹, Sofia Siffo¹, Patricia Papendieck², Rosa Enacan², Laura Grueiro-Papendieck², Ana Chiesa², Héctor M Targovnik¹ and Carina M Rivolta¹*
¹Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Argentina
²División de Endocrinología, Hospital de Nios “Ricardo Gutierrez”, Argentina
*Corresponding author: Carina M Rivolta, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Inmunología, Genética y Metabolismo (INIGEM), Facultad de Farmacia y Bioquímica, Hospital de Clínicas “José de San Martín”, Av. Córdoba 2351, Cuarto Piso, Sala 5, C1120AAR - Buenos Aires, Argentina
Received: October 03, 2016; Accepted: October 14, 2016; Published: November 03, 2016
Abstract
Purpose: DUOX2 deficiency is a transient or permanent disorder that results in thyroid dyshormonogenesis. The purpose of this study was to identify and characterize new mutations in the DUOX2 gene in an attempt to increase the understanding of genotype-phenotype correlation for this disorder. The current study summarizes also the spectrum of DUOX2 variations reported to date in the literature.
Methods: Two siblings from an nonconsanguineous family with clinical and biochemical criteria suggestive of transient CH were studied. Single-Strand Conformation Polymorphism (SSCP) analysis and sequencing of DNA of TPO and DUOX2 genes were performed.
Results: Sequencing analysis of DUOX2 gene revealed two inactivating mutations, a novel c.1057_1058delTT mutation (p.F353Pfs*36, father’s mutation) and a possible previously reported c.1275T>G mutation (p.Y425X, mother’s mutation). Consequently, the two siblings carry a compound heterozygous for p.F353Pfs*36/ p.Y425X mutations, whereas the healthy brother is heterozygous for the c.1275T>G mutation and does not carry the c.1057_1058delTT mutation. Up to date, hundred twenty pathogenic variations and functional single nucleotide polymorphisms in the human DUOX2 gene have been reported associated with transient or permanent CH: 78 missense mutations, 11 nonsense mutations, 26 deletions and insertions, and 6 splice site mutations. The transient or persistent variability of the CH phenotype is not directly related to the number of mutant DUOX2 alleles. Pathogenic DUOX2 mutations were identified together with likely pathogenic variants in the TSHR, DUOXA2, Thyroid peroxidase, Thyroglobulin and SLC26A4 genes.
Conclusion: In the present study, we have identified a novel p.F353Pfs*36 mutation in peroxidase like domain of DUOX2 and we have confirmed that total loss of DUOX2 activity by biallelic premature termination codon causes transient CH phenotype.
Keywords: Congenital hypothyroidism; DUOX2 gene; Mutation; Compound heterozygous mutations; Premature stop codon
Introduction
Iodide Organification Defects (IOD) associated with mutations in the TPO (Thyroid Peroxidase), SLC26A4 (solute carrier family 26 member 4, pendrin), DUOX2 (Dual Oxidasa 2) or DUOXA2 (DUOX maturation factor 2) genes are implicated in the pathogenesis of Congenital Hypothyroidism (CH) [1]. IOD is characterized by high levels of serum TG and TSH with simultaneous low levels of circulating thyroid hormones and a positive Perchlorate Descarge Test (PDT), indicating that the iodide is taken up by thyroid cells but it is not incorporated into the TG protein [1,2].
The key enzymatic system involved in the iodide organification process is located on the apical plasma membrane of polarized follicular thyroid cells. TPO catalyzes both the iodination and coupling of hormonogenic tyrosyl residues of TG with an absolute requirement of hydrogen peroxide, which acts as an electron acceptor [3]. H2O2 is generated by a metabolic pathway, involving DUOX1 and DUOX2 [3-5].
PDT is used to distinguish Total Iodide Organification Defect (TIOD) from Partial Thyroid Organification Defect (PIOD). TPO gene mutations in patients with PIOD usually affect a single allele, whereas homozygous or compound-heterozygous TPO mutations are associated to TIOD [1]. Mutations in SLC26A4 gene cause Pendred syndrome characterized by congenital sensorineural hearing loss, goiter with or without hypothyroidism and usually PIOD [1]. Most of patients with mutations in DUOX2 gene are characterized by PIOD. Furthermore, mutations in DUOX2 gene have been associated to transient CH (TCH) or permanent CH (PCH), with a high intra and interfamilial phenotypic variability [2]. Instead, TCH can be caused by iodine excess or deficiency and exposure to maternal antithyroid drugs or transplacental antibodies [6,7]. PCH requires lifelong thyroid hormone replacement.
Most cases of CH associated with alterations in the DUOX2 are caused by either biallelic or monoallelic mutations which lead to relantionships between DUOX2 genotypes and clinical phenotypes extremaly complex [8-10]. Both biallelic and monoallelic DUOX2 mutations could be associated with TCH or PCH [11,12]. Because DUOX2 biallelic mutations commonly are inherited in an autosomal recessive manner, the patients should be homozygous or compound heterozygous for gene mutations and the parents should be carriers of one DUOX2 mutation. Up to date, 121 variations have been identified and characterized throughout the DUOX2 gene [8,9,11-13-47].
In the present study we report a family with transient CH. Screening of DUOX2 gene revealed two inactivating mutations, a novel c.1057_1058delTT mutation (p.F353Pfs*36) and a possible previously documented c.1275T>G mutation (p.Y425X) [19], conforming a new compound heterozygous. Remarkable, as our findings confirm, severe biallelic defects of DUOX2 in transient CH infers compensatory mechanisms in the peroxide supply.
Materials and Methods
Patients
We report 2 siblings of nonconsanguineous healthy parents and affected with congenital hypothyroidism which led to perform genetic testing.
The proband (II-1), born in 2003, was a girl who was detected through neonatal screening with elevated TSH, 32.5 mUI/l blood at 2 days of life (Cut of <10 mUI/L). Thyroid profile at 1 month of age showed TSH: 32 mU/L (reference range: <10), TT4 13 ug/dl, (reference range 6-18) FT4: 1.46 ng/dl (reference range:1-2.6) and TG: 266 ng/ dl (reference range: 30-100) with no circulating autoantibodies (anti- TPO and anti-TG) (Table 1) and eutopic enlarged thyroid gland in the 99mTc scan. These results suggested a defect in thyroid hormone synthesis and so treatment with levothyroxine was started at 25 μg per day and continued since 2.9 years of age. At that time treatment was discontinued for a month in order to reevaluate her thyroid function. Serum TSH, TT4 and FT4 levels were normal and TG slightly elevated (Table 1). A Perchlorate discharge test was performed with 17% of discharge (normal <15%). Thyroid ultrasound was normal. Treatment was restarted and stopped again for a month at 7 years of age. Thyroid profile was normal (Table 1), perchlorate test negative and TG: 51.2 ng/dl (reference range: 6-40). Therefore levothyroxine replacement was not necessary. She is now 12 years old, grows normally, has an adequate school performance, undergoes normal puberty and keeps euthyroid.
Relative
Age
TSH
mUI/L
TT4
mg/dl
FT4
ng/dl
TT3
ng/dl
TG
ng/ml
Anti-TPO /Anti-TG
U/ml
I-1
1.91
6.7
1.13
118
9.1
<10/<20
I-2
1.23
7.7
1.19
113
10
<10/<20
II-1
Screening at 2 days
32.5 (blood)
1 month
evaluation
32
13
1.46
ND
266
<10/<20
2.72 years
Re-evaluation*
5.22
8.2
1.17
ND
41.7
<10/<20
7 years
Re-evaluation*
4.8
7.2
1.7
145
51.7
<10/<20
11 years
evaluation
2.4
7.0
1.06
154
41.0
<10/<20
II-2
2.60
9.5
1.15
182
10.9
<10/<20
II-3
Screening at 2 days
19 (blood)
15 days
evaluation
33
7.9
0.92
260
666
<10/<20
3.32 years
Re-evaluation *
4.87
10.2
1.37
173
41.7
<10/<20
7 years
evaluation
2.06
8.6
1.27
126
17.6
<10/<20
Reference
range
<60 days: 1.6-10,
2-12 months: 1-6.8, > 1 year: 0.5–6.5
<30 days: 6–18,
>1 month: 6-14,
> 17 years: 4.5–12.5
<30 days: 1–2.6,
>1 month: 0.8–2.2,
> 17 years: 0.8-2.0
<30 days: 80-260,
1-12 months: 80-245, > 1 year 80-220
< 30 days: 30-100, >1 month
6-40
<17 years: <20, Adults <35
* Data were obtained after one month of levothyroxine withdrawal.
Table 1: Laboratory data of patients with transient congenital hypothyroidism and DUOX2 defects.
Her brother (II-3), born in 2007, was also detected through neonatal screening, 19 mUI/L at 2 days of life. Thyroid profile at 15 days showed TSH: 33 mU/L, T4: 7.9 μg/dl, FT4: 0.9 ng/dl and TG: 666 ng/dl (Table 1). He started treatment with levothyroxine 25 μg/day, grew and developed normally and was reevaluated with treatment withdrawal for a month at 3.72 years of age. His thyroid function was normal excepting a slightly high TG (Table 1), thyroid gland was normal on ultrasound and the perchlorate discharge test negative. The treatment was stopped. He grew in the 50 th percentile and has a normal high Intelligence Quotient (IQ) at age 6. With 7 years of age he continues euthyroid and thyroglobulin levels have normalized.
The father (I-1), mother (I-2) and the healthy brother (II-2) of the proband had normal thyroid function (Table 1) without symptoms and manifestations suggesting hypothyroidism.
Written informed consent to participate in the clinical and genetic studies was given by both parents and the research project was approved by the Institutional Review Board.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
PCR amplification
Genomic DNA was isolated from peripheral blood leucocytes by the standard Cetyltrimetilammonium Bromide (CTAB) method and stored at -20 °C up to the analysis.
The promotor region and all 17 exons of the human TPO gene including splicing signals and the flanking regions of each intron, and the complete coding sequence of the human DUOX2 gene, along with the flanking regions of each intron were amplified using the primers and PCR conditions reported previously [17,48]. The amplified products were analysed in 2% agarose gel.
Single strand conformation polymorphism (SSCP) analysis
SSCP analysis was used to screen for the presence of mutations in each exon of the TPO and DUOX2 genes and their flanking intronic regions. The gel matrix for SSCP contained 8%, 10% or 12% polyacrylamide (29:1) (Invitrogen, Life Technologies), with or without 10% glycerol. Samples were electrophoresed for 17-43 h at a constant temperature (4 °C). DNA was visualized by silver staining.
DNA sequencing
DUOX2 PCR products showing an aberrant pattern in SSCP analysis were purified by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and directly sequenced using sense- and antisense-specific primers reported previously [17] with the Big Dyedeoxyterminator Cycle Sequencing Kit (Applied Biosystems, Weiterstadt, Germany). The samples were analysed on the 3130xl and 3500xl Genetic Analyzer (Applied Biosystems).
Sequence variants are numbered according to DUOX2 mRNA reference sequences reported in National Center for Biotechnology Information (NCBI), accession number: NM_014080.4. The DUOX2 gene includes 34 exons, being the first non-coding. The ‘A’ of the ATG start codon is denoted as nucleotide +1 being the initiator methionine, the codon 1.
Cloning of wild-type and mutated exons 9 PCR fragments
The amplified fragment corresponding to exon 9 from index patient II-1 was T-A cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). DNA sequencing was performed as described above from wild type and mutant allele clones using the T7 primer.
Results
Promoter and all 17 exons of TPO gene and all 33 coding exons of the DUOX2 gene, along with the flanking intronic sequences, from patient II-1 and healthy controls were screened by SSCP analysis. The analysis of TPO PCR products did not show aberrant bands, suggesting the absence of TPO gene mutations. Whereas, analysis of DUOX2 PCR products showed two different patterns of migration that were not detected in the healthy controls. Sequence analysis of the samples showing the abnormal SSCP patterns revealed two mutations. One of them was a novel heterozygous double thymine deletion at nucleotide positions 1057 and 1058 (c.1057_1058delTT) in exon 10 (Figure 1), resulting in a frameshift at amino acid 353 with a putative Premature Termination Codon (PTC) at 36 codons downstream, located in the exon 11 [p.F353Pfs*36] (Figure 2). The c.1057_1058delTT heterozygous state of index patient II-1 was confirmed by cloning in pGEM-T Easy vector and sequencing of both wild type and mutant alleles (Figure 1). The second mutation was a possible previously documented heterozygous thymine-toguanine transversion at nucleotide position 1275 (c.1275T>G) in exon 12 (Figure 1) which replaces a tyrosine residue at position 425 by a PTC [p.Y425X] (Figure 2). Previously Cortinovis et al. reported the p.Y425X mutation but did not inform what is the nucleotide change that causes the PTC [19]. Two different mutations may generate a stop codon in this position, c.1275T>G (TAT>TAG) or c.1275T>A (TAT>TAA). These findings indicate that index patient II-1 is a compound heterozygous for c.1057_1058delTT/c.1275T>G mutations.
Figure 1: Segregation analysis of the DUOX2 mutated alleles of the index patient II-1 and the family members. Partial sequencing chromatograms of genomic
DNA are shown. The pedigree shows the pattern of inheritance of the mutant DUOX2 alleles. Squares represent males and circles females. Filled symbols denote
affected individuals and half-filled symbols, unaffected heterozygote individuals. The solid symbols indicate the c.1057_1058delTT mutated allele and the hatched
symbols the c.1275T>G mutated allele. Sense strand is shown. Note that exon 10 was sequenced with the corresponding intronic reverse primer and the reverse
and complementary sequence is shown. Arrows denote the position of identified mutations, single chromatogram peaks indicate homozygosity and two overlapping
peaks at the same locus, heterozygosity. The proband is indicated with an arrow. The index patient II-1 and her brother (II-3) were compound heterozygous for
c.1057_1058delTT and c.1275T>G. The father was heterozygous for c.1057_1058delTT and the mother, and her healthy brothers (II-2) were heterozygous for
c.1275T>G. Partial sequencing chromatograms of wild-type (WT) and mutated (c.1057_1058delTT) alleles corresponding to exon 10 from index patient II-1 cloned
into pGEM-T Easy vector are shown.
Sequencing analysis of PCR products of exons 10 and 12 from index patient II-1, his father (I-1), his mother (I-2), his unaffected brother (II-2) and his affected brother (II-3) showed that II-2 and II-3 inherited one copy of the c.1057_1058delTT mutation from his father and one copy of the c.1275T>G mutation from his mother (Figure 1). The healthy brother (II-1) is heterozygous for the c.1275T>G mutation and does not carry the c.1057_1058delTT mutation.
Discussion
Here, we present two children from an unrelated family with TCH due to biallelic mutations in the DUOX2 gene. Both affected members had clinical and biochemical criteria suggestive of CH associated with DUOX2 deficiency: intact iodide trapping, IOD, elevated-serum TSH with high levels of serum TG. Molecular analyses indicated that the affected individuals are either compound heterozygous for c.1057_1058delTT/c.1275T>G mutations.
The DUOX1 and DUOX2 genes encode similar proteins that are inserted in the apical membrane of thyroid follicular cells. The DUOX2 gene (GenBank accession number NT_010194) is located on chromosome 15q15.3 spanning 22 Kb of genomic DNA which include 34 exons, being the first non-coding. The mRNA (GenBank accession number NM_014080) is 6,376 nucleotides long and the preprotein is composed of a putative 25 amino acids signal peptide followed by a 1,523 amino acids polypeptide [3]. DUOX1 gene encodes a homologous protein displaying 83% sequence similarity with DUOX2. The 36 kb DUOX1 gene consists of 35 exons (being the first two noncoding) and gives rise to a protein of 1551 amino acids whose first 21 correspond to the signal peptide [3]. DUOX2 alterations were first described in 2002 [13]. In the literature, Sanger sequencing has been commonly used for DUOX2 mutations identification in both research and clinical studies. Recently, high throughput sequencing has been reported to be a rapid and effective tool for largest DUOX2 mutation screening for patients with or without family history. The DUOX2 mutations identified in our study and in the literature are reviewed in Table 2. The mutational spectrum shows heterogenous alterations dispersed in the exons and introns of the entire gene. To date, molecular research has shown that 121 pathogenic variations and functional single nucleotide polymorphisms have been found to be associated with TCH, SCH or PCH [8,9,11-13-47]. The most frequent events of deleterious DUOX2 mutations were found to be altered proteins with a single amino acid substitution (78 mutations, see Table 2); followed by deletions/insertion (26 mutations, see Table 2) and nonsense mutations (11 mutations, see Table 2) causing premature truncation of the DUOX2 protein. Only six DUOX2 splice site mutations have been reported: c.514-2A>G (g.IVS5-2A>G) [12], c.2335-1G>C (g.IVS18-1G>C) [31,36], c.2655-2A>C (g.IVS20- 2A>C) [17], c.3516-1G>A (g.IVS26-1G>A) [12], c.3693+1G>T (g.IVS28+1G>T) [12] and c.4240-1G>C (g.IVS31-1G>C) [42]. The p.K530X, p.E897K, p.R1110Q, p.L1160del, p.R1334W and p.L1343F mutations are the most frequently identified DUOX2 mutations in Asian population, whereas the frequent mutation p.S965Sfs*30 was found in Caucasian populations.
#
Exon/Intron
Nucleotide alteration
Amino acid alteration
References
1
2
c.34delC
p.L12Wfs*5
42
2
3
c.108G>C
p.Q36H
17
3
3
c.127A>T
p.N43Y
30, 42
4
4
c.184C>T
p.P62S
38
5
4
c.214G>T
p.A72S
30
6
4
c.244C>A
p.R82S
11
7
4
c.244C>T
p.R82C
12
8
4
c.287C>T
p.P96L
30
9
4
c.298A>G
p.N100D
9
10
5
c.343G>T
p.D115Y
41
11
5
c.377-379delCCG
p.A126del
12
12
5
c.398T>A
p.I133N
42
13
5
c.413C>T
p.P138L
31, 41
14
5
c.477delC
p.P159Pfs*17
12
15
5
c.512T>C
p.L171P
31
16
Intron 5
c.514-2A>G (g.IVS5-2A>G)
12
17
6
c.518A>G
p.N173S
40
18
6
c.534G>T
p.W178C
12
19
6
c.596delC
p.S199Wfs*122
12, 41
20
6
c.602_603insG
skipping of exon 5
p.G201Gfs*100
p.A172Pfs*82
12, 16, 31
21
6
c.605_621delAGCTGGCGTCGGGGCCC
p.Q202Rfs*93
12, 30, 42
22
6
c.616G>T
p.G206V
30
23
6
c.647-656delAGAACCCCCTins15 (NA)
p.Q216Lfs*107
12
24
7
c.804delG
p.R268Sfs*53
41
25
8
c.903G>T
p.W301C
11, 34
26
8
c.908C>G
p.P303R
31, 40
27
9
c.979G>T
p.E327*
12, 26
28
9
c.1021C>T
p.P341S
31
29
10
c.1057_1058delTT
p.F353Pfs*36
Present study
30
10
c.1060C>T
p.R354W
27, 39, 43
31
10
c.1097C>G
p.A366G
42
32
10
c.1126C>T
p.R376W
14, 19, 24
33
11
c.1219G>T
p.V407F
44
34
11
c.1232G>A
p.R411K
42
35
12
NA
p.W414*
19
36
12
c.1253delG
p.G418Afs*65
17
37
12
c.1275T>G
p.Y425*
Present study, 19
38
12
c.1300C>T
p.R434*
13, 29
39
13
c.1424A>G
p.Y475C
19
40
13
c.1435_1440delCTATCCinsAG
p.L479Sfs*3
20, 28, 41
41
13
c.1462G>A
p.G488R
25, 28, 30, 44
42
13
c.1516G>A
p.D506N
16, 19
43
13
c.1537G>A
p.D513N
42
44
13
c.1564delA
p.T522Pfs*64
31
45
14
c.1588A>T
p.K530*
11, 12, 20, 34, 41
46
14
c.1621C>T
p.R541W
42
47
15
c.1709A>T
p.Q570L
27, 31, 43
48
15
c.1736T>C
p.L579P
11, 34
49
16
c.1871delG
p.G624Afs*15
25
50
16
c.1883delA
p.K628Rfs*11
12, 20, 41, 44
51
16
c.1931A>G
p.K644R
38
52
17
c.1946C>A
p.A649E
20, 41, 44
53
17
c.2000delT
p.L667Rfs*9
44
54
17
c.2033A>G
p.H678R
8, 20, 25, 26, 30, 31, 32, 33, 41
55
17
c.2048G>T
p.R683L
11, 34, 37, 38
56
17
c.2056C>T
p.Q686*
13, 27, 43
57
17
c.2101C>T
p.R701*
9, 12, 13
58
17
c.2102G>A
p.R701Q
8, 31
59
17
c.2102_2104delGAG
p.G702del
11
60
17
c.2146delC
p.L716Wfs*34
25
61
18
c.2182G>A
p.A728T
8
62
18
c.2202G>A
p.W734*
9
63
18
c.2203G>A
p.D735N
42
64
18
c.2290C>T
p.R764W
43
65
18
c.2303T>C
p.L768P
12
66
Intron 18
c.2335-1G>C (g.IVS18-1G>C)
31, 36
67
19
c.2335G>A
p.V779M
38, 41, 44
68
19
c.2377_2378dupGA
p.D793Efs*11
25
69
19
c.2524C>T
p.R842*
11, 14, 19, 34
70
20
c.2597T>G
p.M866R
31
71
20
c.2635G>A
p.E879K
11, 12, 20, 30, 34, 41, 44
72
20
c.2654G>A
p.R885Q
20, 30, 41, 44, 45, 47
73
20
c.2654G>T
p.R885L
25, 38, 44
74
Intron 20
c.2655-2A>C (g.IVS20-2A>C)
17
75
21
c.2732C>T
p.S911L
22
76
21
c.2843delG
p.G948Vfs*48
41
77
22
c.2895_2898delGTTC
p.S965Sfs*30
8, 13, 15, 17, 19, 31, 43
78
22
c.2894C>T
p.S965L
38
79
23
c.2944C>G
p.P982A
8, 31
80
24
c.3076C>T
p.Q1026*
15
81
24
c.3082_3085delCTGCinsGCTTCCT
p.L1028Afs*3
43
82
24
c.3096G>C
p.K1032N
41
83
24
c.3116G>A
p.R1039Q
42
84
24
c.3155G>A
p.C1052Y
22, 31
85
24
c.3179C>T
p.A1060V
44
86
25
c.3200C>T
p.S1067L
20, 31, 41
87
25
c.3239T>C
p.I1080T
41, 25, 44
88
25
c.3251G>A
p.R1084Q
11
89
25
c.3264_3267delCAGC
p.A1088Afs*18
36
90
25
c.3329G>A
p.R1110Q
9, 11, 12, 21, 25, 30, 34, 38, 41, 42, 44, 46
Table 2: Spectrum of DUOX2 mutations.
91
25
c.3340delC
p.L1114Sfs*56
11, 34
92
25
c.3367G>A
p.A1123T
12, 30
93
25
c.3383G>A
p.R1128H
44
94
25
c.3391G>T
p.A1131S
9, 11
95
25
c.3413C>A
p.A1138D
11, 34
96
26
c.3449A>G
p.Y1150C
8
97
26
c.3478_3480delCTG
p.L1160del
11, 25, 41, 42, 44, 46
98
Intron 26
c.3516-1G>A (g.IVS26-1G>A)
12
99
Intron 26-exon 34
c.3516-?_4647+?del
p.G1172_F1548del
23
100
27
c.3516-3531delGTCCAAGCTTCCCCAG
p.G1172Gfs*14
12
101
27
c.3540T>A
p.Y1180*
42, 46
102
27
c.3541T>G
p.W1181G
9
103
28
c.3616G>A
p.A1206T
9, 39, 44
104
28
c.3632G>A
p.R1211H
33
105
28
c.3652T>C
p.F1218L
12
106
Intron 28
c.3693+1G>T (g.IVS28+1G>T)
12
107
29
c.3799C>T
p.R1267W
9
108
30
c.3967G>A
p.A1323T
11, 35
109
30
c.4000C>T
p.R1334W
11, 12, 30, 34, 46
110
30
c.4027C>T
p.L1343F
11, 34, 35, 37, 38, 41
111
30
c.4040A>G
p.Y1347C
32
112
31
c.4171C>G
p.P1391A
44
113
31
c.4176T>G
p.F1392L
12
114
Intron 31
c.4240-1G>C (g.IVS31-1G>C)
42
115
32
c.4334T>A
p.V1445E
44
116
32
c.4348T>C
p.Y1450H
44
117
33
c.4405G>A
p.E1469K
44
118
33
c.4408C>T
p.R1470W
38
119
33
c.4475G>A
p.R1492H
11, 41
120
34
c.4552G>A
p.G1518S
23
121
34
c.4637A>G
p.E1546G
31
Sequence variants are numbered according to DUOX2 mRNA reference sequences reported in National Center for Biotechnology Information (NCBI), accession number:NM_014080.4. The DUOX2 gene includes 34 exons, being the first non-coding. The ‘A’ of the ATG start codon is denoted as nucleotide +1 being the initiator methionine, the codon 1. Splicing mutations are annotated by using cDNA sequences and old nomenclature (g.IVS) is included. Frameshifting mutations are designated by “fs” and stop codón with “*”, the length of the shifted open reading frame include the first affected amino acid. NA: Not Available.
Table 2 & off:
The structure of DUOX2 protein contains three regions: 1) peroxidase homology ectodomain (peroxidase-like domain): an amino-terminal extracellular region of approximately 600 residues, which has 43% sequence homology with hemoperoxidasas, which distinguishes DUOX from other family NOX/DUOX, 2) a central region encompassing an intracellular loop containing potentially two EF-hand calcium binding motifs, which would be involved in regulating the conformation and activity of NADPH oxidase; 3) gp91phox/NOX2-like domain: a carboxy-terminal region referred to as the comprising the last six transmembrane domains [3-5,48,49] (Figure 2). These six alpha helices include four conserved histidines, considered two coordination sites of prosthetic group of type heme. The C-terminal cytosolic orientation presents a binding site for the FAD and NADPH [3-5,49]. It is believed that this region is responsible for the transfer of electrons from NADPH through the membrane. The c.1057_1058delTT mutation results in a putative truncated protein of 388 amino acids (Figure 2). The functional consequence of the PTC in p.F353Pfs*36 and p.Y425X mutated proteins, could be the complete ability loss to generate thyroid H2O2. Both PTCs eliminate the gp91phox/NOX2-like domain, and partially the peroxidase-like domain (Figures 2). In addition to altered DUOX2 by truncated proteins as a pathophysiological mechanism in the generation of congenital hypothyroidism, the mutated mRNAs generated in our patient could represent adequate targets for Nonsense Mediated mRNA decay (NMD) pathway, a known RNA surveillance mechanism that detects and rapidly degrades selectively mRNAs that contains PTCs and this could also contribute to the development of the disease [50]. Transcripts from genes carrying PTCs are subject to extreme downregulation, often by 10- to 100-fold and sometimes a complete degradation of mutant mRNA occurs. Specifically, when at least one intron downstream from the PTC and a minimal distance of 50 to 55 nucleotides between the PTC and the final intron, the mRNA is considered defective and is subjected to NMD pathway. The distance between the PTC in p.F353Pfs*36 mutated protein and the start of intron 11 is of 69 nucleotides, whereas the distance between PTC in p.Y425X mutated protein and the start of intron 12 is of 123 nucleotides. Therefore, the position of both PTCs in our study satisfies the condition in order to NMD pathway can be activated.
Figure 2: Structural organization of the wild-type and putative mutant DUOX2 proteins. The peroxidase-like and gp91phox/NOX2-like domains, glycosilation sites
(NXS/T site), EF-hand calcium motifs, and, FAD and NADPH binding sites, drawn to scale, are shown. The seven alpha helice transmembrane domains are
represented by boxes. The partial nucleotide and the deduced amino acid sequences from wild-type are reported below of the respective schematic protein
diagrams. The nucleotide sequence is given in the upper line, and the amino acid translation (represented by single-letter code) is given below their respective
codons. The arrows denote the positions of the c.1057_1058delTT and c.1275T>G mutations and are boxed in the wild-type sequence. The resulting frameshift due
to c.1057_1058delTT mutation is underlined. The nucleotide position in human DUOX mRNA is designated according to reference sequences (NCBI, accession
number: NM_014080.4). The ‘A’ of the ATG start codon is denoted as nucleotide +1 being the initiator methionine, the codon 1. Indicates exon/exon boundaries
and exon numbering is shown.
CH is the most frequent endocrine disease in the infant and one of the most common preventable causes of cognitive and motor impairment. The incidence of CH has increased in recent years with a current prevalence of 1:1400 to 1:2800 [51]. This increase is mainly due to the widespread lowering of TSH screening cutoffs, which has led to detecting mildest forms of CH with normally positioned thyroid gland [52,53]. Whereas the majority of the cases of CH are due to dysembryogenesis or dysgenesis of the thyroid gland (agenesis, ectopia, hypoplasia), one fifth of these hypothyroid newborns have a defect in genes involved in thyroid hormone biosynthesis (dyshormonogenesis) [1]. The prevalence of DUOX2 mutations in thyroid dyshormonogenesis is estimated to be in the range of 29- 45 % [8,13,25,31]. In Asian, the prevalence of DUOX2 mutations in thyroid dyshormonogenesis is higher than in Caucasians [38]. Recent studies were carried out from Chinese patients with CH in order to establish a genotype-phenotype correlation [11,12,34,37]. The genotype-phenotype analysis of 131 Chinese patients with CH is summarized in Table 3. So, one or two DUOX2 mutations were associated with SCH or TCH, whereas patients with three or more DUOX2 pathogenic variants were mostly associated with PCH (Table 3). In addition, compound DUOX2 mutations with other thyroid genes have been identified: TSHR [30,35,38,44], DUOXA2 [45,47], TPO [37,38,42,46], TG [11,38,43] and SLC26A4 [38] genes. However, to date are no functional evidence that the addictive effect of digenic mutations contribute to the CH phenotype.
# of DUOX2
mutations
# of patients
TCH
PCH or APCH
SCH or MPCH
ND
1
76
51 (67 %)
7 (9 %)
15 (20 %)
3 (4 %)
2
37
27 (73 %)
2 (5,4 %)
3 (8.1 %)
5 (13.5 %)
3
16
2 (12.5 %)
14 (87.5 %)
0
0
4
1
0
1 (100 %)
0
0
5
1
0
1 (100 %)
0
0
Total
131
80 (61.1 %)
25 (19.1 %)
18 (13.7 %)
8 (6.1 %)
TCH: Transient Congenital Hypothyroidism; PCH: Permanent Congenital Hypothyroidism; APCH: Apparent Permanent Congenital Hypothyroidism; SCH: Subclinical Congenital Hypothyroidism; MPCH: Mild Permanent Congenital Hypothyroidism; NA: Not Available.
Table 3: Genotype-phenotype correlation in 131 Chinese patients with congenital hypothyroidism with DUOX2 variants [11,12,34,37].
In conclusion, the present study clearly established that total loss of DUOX2 activity by PTC causes TCH phenotype. Our findings also provide further evidence that the transient or persistent variability of the CH phenotype is not directly related to the number of mutant DUOX2 alleles, suggesting the existence of ethnic differences, additional genetic and epigenetic changes, environmental/ geographical factors and the existence of additional H2O2 supply provided by DUOX1, which play a role in the determining severity of this type of form of thyroid dyshormonogenesis.
Acknowledgement
F.S. Belforte is research fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
S. Siffo is research fellows of the Fondo para la Investigación Científica y Tecnológica (FONCyT-ANPCyT-MINCyT).
H.M. Targovnik and C.M. Rivolta are established investigators of the CONICET.
This study was funded by grants from the FONCyT-ANPCyTMINCyT (PICT 2014-1193 to CMR, PICT 2012-1090 and PICT 2015- 1811 to HMT), CONICET (PIP 2015-11220150100499 to CMR) and Universidad de Buenos Aires (UBACyT 2016-20020150100099BA to CMR).
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