- Email: [email protected]
TGM6 gene mutations in undiagnosed cerebellar ataxia patients
Accepted Manuscript TGM6 gene mutations in undiagnosed cerebellar ataxia patients Zhi-hua Yang, Meng-meng Shi, Yu-tao Liu, Yan-lin Wang, Hai-yang Luo, Zhi-lei Wang, Chang-he Shi, Yu-ming Xu PII:
S1353-8020(17)30234-1
DOI:
10.1016/j.parkreldis.2017.07.001
Reference:
PRD 3346
To appear in:
Parkinsonism and Related Disorders
Received Date: 1 March 2017 Revised Date:
29 June 2017
Accepted Date: 3 July 2017
Please cite this article as: Yang Z-h, Shi M-m, Liu Y-t, Wang Y-l, Luo H-y, Wang Z-l, Shi C-h, Xu Y-m, TGM6 gene mutations in undiagnosed cerebellar ataxia patients, Parkinsonism and Related Disorders (2017), doi: 10.1016/j.parkreldis.2017.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
TGM6 gene mutations in undiagnosed cerebellar ataxia patients Zhi-hua Yang, MD1, Meng-meng Shi, MD1, Yu-tao Liu, MD, PhD1, Yan-lin Wang,
Xu, MD, PhD1*
RI PT
MD1, Hai-yang Luo, MD1, Zhi-lei Wang, MD1, Chang-he Shi, MD, PhD1*, Yu-ming
1 Department of Neurology, The first affiliated Hospital of Zhengzhou University,
SC
Zhengzhou University, Zhengzhou, 450000, Henan, China.
M AN U
* Corresponding Author: Yu-ming Xu & Chang-he Shi
Address: Department of Neurology, The first affiliated Hospital of Zhengzhou University, Zhengzhou University.
1 Jian-she east road, Zhengzhou 450000, Henan, China
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Tel: +86-371-66862132, Fax: +86-371-66862132
E-mail: [email protected] & [email protected]
EP
Potential conflict of interest: None.
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Acknowledgements: The work was supported by grant U1404311 from the National Natural Science Foundation of China (to Dr Changhe Shi) and grants 81530037 and 81471158 from the National Natural Science Foundation of China (to Dr Yuming Xu).
Key words： ：Spinocerebellar ataxias；SCA35；Mutations
ACCEPTED MANUSCRIPT Autosomal dominant spinocerebellar ataxias (SCAs) are the most common types of hereditary cerebellar ataxia. Mutations in TGM6 have been identified as the cause of SCA35 in several Chinese SCA families. The main clinical manifestations include
RI PT
a slowly progressive course, trunk/limb ataxia, and hand tremors.[1-3]. However, no TGM6 mutations in other populations or in individuals with sporadic cerebellar ataxia
SC
were reported[4].
In this study, we performed comprehensive mutation screening of TGM6 by
M AN U
Sanger sequencing in a cohort of undiagnosed cerebellar ataxia patients, including 75 probands of autosomal dominant SCA families and 102 patients with sporadic cerebellar ataxia. All patients were followed in the Department of Neurology in the First Affiliated Hospital of Zhengzhou University. Meanwhile, 200 healthy Chinese
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individuals were analyzed as ethnically matched controls. The study was approved by the Ethics Committee of First Affiliated Hospital of Zhengzhou University. All
EP
subjects in our research were Chinese and provided written informed consent.
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We identified two mutations of TGM6 in these patients, including a novel splice-site mutation in a SCA family (family1) and a de novo missense mutation in a sporadic cerebellar ataxia patient (family2). In family 1(Fig.1A), the proband (Ⅲ-4) presented with slowly progressive gait unsteadiness, dysarthria, and hand tremor with onset of symptoms at age 54. Her sister (Ⅲ-3) had been experiencing similar symptoms since age 59. Their deceased father had suffered from progressive gait disturbance and dysarthria beginning at age 50 and had died following a stroke at age
ACCEPTED MANUSCRIPT 52. Other members of the family (Ⅲ-1, Ⅲ-2, Ⅲ-5, Ⅱ-1, and Ⅱ-4) were without neurological symptoms. MRI of the brain in Ⅲ-3 and Ⅲ-4 demonstrated diffuse cerebellar atrophy; MRI was normal in Ⅲ-5 (Fig.1B). In family 2 (Fig.1A), the
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proband was a 62-year-old female patient (Ⅱ-1) who presented with progressive gait unsteadiness, dysarthria, hand tremor, and memory impairment that had first appeared at age 60. MRI of the brain demonstrated mild cerebellar atrophy (Figure not shown).
SC
However, other family members (I-1, I-2 andⅡ-2) were all healthy, without any
M AN U
neuropsychological dysfunction .
A splice-site mutation in TGM6, c.7+1G>T, was identified in the proband (Ⅲ4) of family 1 (Fig.1C). Subsequently, Sanger sequencing in other family members (Ⅱ-1, Ⅱ-4, Ⅲ-1, Ⅲ-2, Ⅲ-3, and Ⅲ-5) confirmed that the other patient (Ⅲ-3) also
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harbored the mutation, and that no clinically unaffected family members carried the mutation except for a 44-year-old pre-symptomatic carrier (Ⅲ-5). The mutation was
EP
absent in 200 ethnically matched controls. In family 2, mutation screening for TGM6 showed a missense mutation c.1478C>T, p.P493L in the proband (Ⅱ-1) (Fig.1C),
AC C
while other family members (I-1, I-2 andⅡ-2) in the family and 200 healthy controls were without the mutation.
Transglutaminase 6 (TGM6, NM_198994) encodes transglutaminase 6 protein
(TG6), which is a member of the transglutaminase family of Ca2+ dependent enzymes that catalyze the cross-linking of proteins and the conjugation of polyamines to proteins[5]. Previous functional studies and bioinformatics analysis have revealed that
ACCEPTED MANUSCRIPT TGM6 is associated with ataxia and that mutation in TGM6 may decrease TG6 stability and transglutaminase activity[1-3]. In this study, the mutation c.7+1G>T affected the initial nucleotide of the splice donor site of intron 1 (IVS+1G>T). Several
RI PT
splice-site mutation bioinformatics tools, including HSF (Human Splicing Finder), SSS (Splice site score calculation), and Max EntScan, revealed decreased splicing ability of the splice-site mutation, which might influence the normal splicing of
SC
mRNA and further damage the physiological functions of TG6. The mutation
M AN U
c.1478C>T, p.P493L affected conserved amino acids（Fig.1D）. Using I-Mutant, the mutation would decrease the stability of TG6. Using AGGRESCAN, the wild type 493P was located at a hotspot area (aggregation-prone segment) from the 496th to the 503th site, and the mutant type 493L extended this area to involve the 494th site to the
TE D
503th site, which might also change the normal protein folding.
In summary, we identified a novel splice-site mutation of TGM6 in a SCA family，
EP
and a de novo missense mutation of TGM6 in a sporadic cerebellar ataxia patient. Our study illustrates the importance of TGM6 mutations in Chinese cerebellar ataxia
AC C
patients, not only in SCA families, but also in individuals with sporadic cerebellar ataxia. Genetic testing of SCA35 should be considered for undiagnosed cerebellar ataxia patients in clinical practice.
Disclosure
The authors report no conflicts of interest relevant to the manuscript.
Acknowledgements
ACCEPTED MANUSCRIPT The work was supported by grant U1404311 from the National Natural Science Foundation of China (to Dr Changhe Shi) and grants 81530037 and 81471158 from the National Natural Science Foundation of China (to Dr Yuming Xu).
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References
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M AN U
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[1] Wang JL, Yang X, Xia K, Hu ZM, Weng L, Jin X, et al. TGM6 identified as a novel causative gene of spinocerebellar ataxias using exome sequencing. Brain : a journal of neurology. 2010;133:3510-8. [2] Li M, Pang SY, Song Y, Kung MH, Ho SL, Sham PC. Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in a Chinese family. Clinical genetics. 2013;83:269-73. [3] Guo YC, Lin JJ, Liao YC, Tsai PC, Lee YC, Soong BW. Spinocerebellar ataxia 35: novel mutations in TGM6 with clinical and genetic characterization. Neurology. 2014;83:1554-61. [4] Fogel BL, Lee JY, Lane J, Wahnich A, Chan S, Huang A, et al. Mutations in rare ataxia genes are uncommon causes of sporadic cerebellar ataxia. Movement disorders : official journal of the Movement Disorder Society. 2012;27:442-6. [5] Grenard P, Bates MK, Aeschlimann D. Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z. The Journal of biological chemistry. 2001;276:33066-78.
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Fig1. The pedigree charts, genetic findings and brain MRI in the family1 and faimly2. A: Pedigree chart: asterisk, members sequenced. B: T2-weighted brain MRI
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of Ⅲ-3, Ⅲ-4 and Ⅲ-5 in family1. C: Sequencing chromatograms. D: Conservation analysis of the mutation site，493P.
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SC
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ACCEPTED MANUSCRIPT
S1353-8020(17)30234-1
DOI:
10.1016/j.parkreldis.2017.07.001
Reference:
PRD 3346
To appear in:
Parkinsonism and Related Disorders
Received Date: 1 March 2017 Revised Date:
29 June 2017
Accepted Date: 3 July 2017
Please cite this article as: Yang Z-h, Shi M-m, Liu Y-t, Wang Y-l, Luo H-y, Wang Z-l, Shi C-h, Xu Y-m, TGM6 gene mutations in undiagnosed cerebellar ataxia patients, Parkinsonism and Related Disorders (2017), doi: 10.1016/j.parkreldis.2017.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
TGM6 gene mutations in undiagnosed cerebellar ataxia patients Zhi-hua Yang, MD1, Meng-meng Shi, MD1, Yu-tao Liu, MD, PhD1, Yan-lin Wang,
Xu, MD, PhD1*
RI PT
MD1, Hai-yang Luo, MD1, Zhi-lei Wang, MD1, Chang-he Shi, MD, PhD1*, Yu-ming
1 Department of Neurology, The first affiliated Hospital of Zhengzhou University,
SC
Zhengzhou University, Zhengzhou, 450000, Henan, China.
M AN U
* Corresponding Author: Yu-ming Xu & Chang-he Shi
Address: Department of Neurology, The first affiliated Hospital of Zhengzhou University, Zhengzhou University.
1 Jian-she east road, Zhengzhou 450000, Henan, China
TE D
Tel: +86-371-66862132, Fax: +86-371-66862132
E-mail: [email protected] & [email protected]
EP
Potential conflict of interest: None.
AC C
Acknowledgements: The work was supported by grant U1404311 from the National Natural Science Foundation of China (to Dr Changhe Shi) and grants 81530037 and 81471158 from the National Natural Science Foundation of China (to Dr Yuming Xu).
Key words： ：Spinocerebellar ataxias；SCA35；Mutations
ACCEPTED MANUSCRIPT Autosomal dominant spinocerebellar ataxias (SCAs) are the most common types of hereditary cerebellar ataxia. Mutations in TGM6 have been identified as the cause of SCA35 in several Chinese SCA families. The main clinical manifestations include
RI PT
a slowly progressive course, trunk/limb ataxia, and hand tremors.[1-3]. However, no TGM6 mutations in other populations or in individuals with sporadic cerebellar ataxia
SC
were reported[4].
In this study, we performed comprehensive mutation screening of TGM6 by
M AN U
Sanger sequencing in a cohort of undiagnosed cerebellar ataxia patients, including 75 probands of autosomal dominant SCA families and 102 patients with sporadic cerebellar ataxia. All patients were followed in the Department of Neurology in the First Affiliated Hospital of Zhengzhou University. Meanwhile, 200 healthy Chinese
TE D
individuals were analyzed as ethnically matched controls. The study was approved by the Ethics Committee of First Affiliated Hospital of Zhengzhou University. All
EP
subjects in our research were Chinese and provided written informed consent.
AC C
We identified two mutations of TGM6 in these patients, including a novel splice-site mutation in a SCA family (family1) and a de novo missense mutation in a sporadic cerebellar ataxia patient (family2). In family 1(Fig.1A), the proband (Ⅲ-4) presented with slowly progressive gait unsteadiness, dysarthria, and hand tremor with onset of symptoms at age 54. Her sister (Ⅲ-3) had been experiencing similar symptoms since age 59. Their deceased father had suffered from progressive gait disturbance and dysarthria beginning at age 50 and had died following a stroke at age
ACCEPTED MANUSCRIPT 52. Other members of the family (Ⅲ-1, Ⅲ-2, Ⅲ-5, Ⅱ-1, and Ⅱ-4) were without neurological symptoms. MRI of the brain in Ⅲ-3 and Ⅲ-4 demonstrated diffuse cerebellar atrophy; MRI was normal in Ⅲ-5 (Fig.1B). In family 2 (Fig.1A), the
RI PT
proband was a 62-year-old female patient (Ⅱ-1) who presented with progressive gait unsteadiness, dysarthria, hand tremor, and memory impairment that had first appeared at age 60. MRI of the brain demonstrated mild cerebellar atrophy (Figure not shown).
SC
However, other family members (I-1, I-2 andⅡ-2) were all healthy, without any
M AN U
neuropsychological dysfunction .
A splice-site mutation in TGM6, c.7+1G>T, was identified in the proband (Ⅲ4) of family 1 (Fig.1C). Subsequently, Sanger sequencing in other family members (Ⅱ-1, Ⅱ-4, Ⅲ-1, Ⅲ-2, Ⅲ-3, and Ⅲ-5) confirmed that the other patient (Ⅲ-3) also
TE D
harbored the mutation, and that no clinically unaffected family members carried the mutation except for a 44-year-old pre-symptomatic carrier (Ⅲ-5). The mutation was
EP
absent in 200 ethnically matched controls. In family 2, mutation screening for TGM6 showed a missense mutation c.1478C>T, p.P493L in the proband (Ⅱ-1) (Fig.1C),
AC C
while other family members (I-1, I-2 andⅡ-2) in the family and 200 healthy controls were without the mutation.
Transglutaminase 6 (TGM6, NM_198994) encodes transglutaminase 6 protein
(TG6), which is a member of the transglutaminase family of Ca2+ dependent enzymes that catalyze the cross-linking of proteins and the conjugation of polyamines to proteins[5]. Previous functional studies and bioinformatics analysis have revealed that
ACCEPTED MANUSCRIPT TGM6 is associated with ataxia and that mutation in TGM6 may decrease TG6 stability and transglutaminase activity[1-3]. In this study, the mutation c.7+1G>T affected the initial nucleotide of the splice donor site of intron 1 (IVS+1G>T). Several
RI PT
splice-site mutation bioinformatics tools, including HSF (Human Splicing Finder), SSS (Splice site score calculation), and Max EntScan, revealed decreased splicing ability of the splice-site mutation, which might influence the normal splicing of
SC
mRNA and further damage the physiological functions of TG6. The mutation
M AN U
c.1478C>T, p.P493L affected conserved amino acids（Fig.1D）. Using I-Mutant, the mutation would decrease the stability of TG6. Using AGGRESCAN, the wild type 493P was located at a hotspot area (aggregation-prone segment) from the 496th to the 503th site, and the mutant type 493L extended this area to involve the 494th site to the
TE D
503th site, which might also change the normal protein folding.
In summary, we identified a novel splice-site mutation of TGM6 in a SCA family，
EP
and a de novo missense mutation of TGM6 in a sporadic cerebellar ataxia patient. Our study illustrates the importance of TGM6 mutations in Chinese cerebellar ataxia
AC C
patients, not only in SCA families, but also in individuals with sporadic cerebellar ataxia. Genetic testing of SCA35 should be considered for undiagnosed cerebellar ataxia patients in clinical practice.
Disclosure
The authors report no conflicts of interest relevant to the manuscript.
Acknowledgements
ACCEPTED MANUSCRIPT The work was supported by grant U1404311 from the National Natural Science Foundation of China (to Dr Changhe Shi) and grants 81530037 and 81471158 from the National Natural Science Foundation of China (to Dr Yuming Xu).
RI PT
References
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M AN U
SC
[1] Wang JL, Yang X, Xia K, Hu ZM, Weng L, Jin X, et al. TGM6 identified as a novel causative gene of spinocerebellar ataxias using exome sequencing. Brain : a journal of neurology. 2010;133:3510-8. [2] Li M, Pang SY, Song Y, Kung MH, Ho SL, Sham PC. Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in a Chinese family. Clinical genetics. 2013;83:269-73. [3] Guo YC, Lin JJ, Liao YC, Tsai PC, Lee YC, Soong BW. Spinocerebellar ataxia 35: novel mutations in TGM6 with clinical and genetic characterization. Neurology. 2014;83:1554-61. [4] Fogel BL, Lee JY, Lane J, Wahnich A, Chan S, Huang A, et al. Mutations in rare ataxia genes are uncommon causes of sporadic cerebellar ataxia. Movement disorders : official journal of the Movement Disorder Society. 2012;27:442-6. [5] Grenard P, Bates MK, Aeschlimann D. Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z. The Journal of biological chemistry. 2001;276:33066-78.
EP
Fig1. The pedigree charts, genetic findings and brain MRI in the family1 and faimly2. A: Pedigree chart: asterisk, members sequenced. B: T2-weighted brain MRI
AC C
of Ⅲ-3, Ⅲ-4 and Ⅲ-5 in family1. C: Sequencing chromatograms. D: Conservation analysis of the mutation site，493P.
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SC
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ACCEPTED MANUSCRIPT