Pediatric Neurology 53 (2015) 262e265
Contents lists available at ScienceDirect
Pediatric Neurology journal homepage: www.elsevier.com/locate/pnu
Clinical Observations
Two Novel Heterozygous Mutations in ERCC8 Cause Cockayne Syndrome in a Chinese Patient Yun-pu Cui MD a, Yi-yu Chen MMed b, c, Xue-mei Wang MD a, Xin-li Wang MD, PhD a, *, Xu Nan BS c, d, Hongshan Zhao PhD c, d, * a
Department of Pediatrics, Peking University Third Hospital, Beijing, China Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing, China c Human Disease Genomics Center, Peking University, Beijing, China d Department of Medical Genetics, School of Basic Medical Sciences, Peking University, Beijing, China b
abstract BACKGROUND: Cockayne syndrome (MIM #133540, Cockayne syndrome B; 216400, Cockayne syndrome A) is a rare
autosomal recessive inherited disease in which the characteristic symptoms are premature aging, cachectic dwarfism, lack of subcutaneous fat, neurological alterations, light sensitivity, and failure to thrive. The mutated gene responsible for this syndrome has been identified as usually either CSA (CKN1, ERCC8) or CSB (ERCC6). In this study, we describe the case of a 7-year-old Chinese boy with characteristic symptoms of Cockayne syndrome A and the conduction of mutation screening of the CSA gene. METHODS: The patient was diagnosed with Cockayne syndrome in the pediatrics clinic for growth failure and developmental delay. We collected peripheral blood samples of the patient and his parents and then extracted the genomic DNA. DNA samples from control subjects and the patient were subjected to polymerase chain reaction amplification. All exons and the flanking intron-exon boundaries of CSA were amplified; then, the polymerase chain reaction products were directly sequenced for mutation screening. RESULTS: Two novel heterozygous CSA mutations, c.551-2A>C and c.394_398delTTACA, were identified in the patient. The c.551-2A>C mutation originates from his father and changed the splice acceptor site AG to CG, thus possibly causing alternative splicing. The c.394_398delTTACA from his mother caused a frameshift after the amino acid at position 132, thus introducing a premature stop codon in the gene sequence. CONCLUSIONS: These mutations extend the mutation spectrum of Cockayne syndrome in the context of Chinese race and provide possibilities of prenatal diagnosis for future offsprings in this family. Keywords: Cockayne syndrome, CSA, autosomal recessive, mutation
Pediatr Neurol 2015; 53: 262-265 Ó 2015 Elsevier Inc. All rights reserved.
Cockayne syndrome (MIM #133540, 216400) is a rare autosomal recessive inherited disease in which the characteristic symptoms are premature aging, cachectic dwarfism, lack of subcutaneous fat, neurological alterations, light sensitivity, and failure to thrive. Since first described by Cockayne in 1936,1 about 150-200 cases have been reported worldwide, and the mutated gene responsible for Y.-p.C. and Y.-y.C. contributed equally to this work. Conflict of interest: Nothing to declare.
Article History: Received November 26, 2013; Accepted in final form March 15, 2014 * Communications should be addressed to: Dr. Wang; Department of Pediarics; Peking University Third Hospital; Dr. Zhao; Department of Medical Genetics; Peking University Health Science Center; Human Disease Genomics Center; Peking University; Beijing, China. E-mail addresses: [email protected], [email protected] 0887-8994/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pediatrneurol.2015.06.006
this syndrome has been identified as usually either CSA (CKN1, ERCC8)2 or CSB (ERCC6).3 Cockayne syndrome, xeroderma pigmentosum (MIM #278700, 278720, 278730, 278740, 278760, 278780, 610651), and trichothiodystrophy (MIM #601675) constitute the nucleotide excision repair (NER) disorder family.4 The role that CSA protein plays in NER is revealed: when the elongating RNAPIIo encounters DNA lesions, the RNA polymerase II (RNAPIIo) is stalled and this triggers the recruitment of the NER proteins; CSA is dispensable for the attraction of NER proteins, whereas cooperation with CSB is required to recruit XPA binding protein 2 (XAB2), high mobility group nucleosome binding domain 1 (HMGN1), and transcription elongation factor A (SII)(TFIIS); and CSB serves as a repair coupling factor to recruit other NER proteins, chromatin remodelers, and CSAE3-ubiquitin ligase complexes to the stalled RNAPIIo.5,6
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
It is reported that the incidence of Cockayne syndrome in western European is 2.7 per million.7 However, there were no incidences, and only one mutation report of CSB was found in the Chinese population until now. In this article, we report a new paternal splice acceptor site mutation (c.551-2A>C) and a maternal five-nucleotide deletion mutation (c.394_398delTTACA) found in a Chinese family. These mutations extend the mutation spectrum of Cockayne syndrome in the context of Chinese race and provide possibilities of prenatal diagnosis for future offsprings in this family. Materials and Methods Patient and control samples
The patient is a 7-year-old boy of Han nationality who was the firstborn child of nonconsanguineous parents. He was brought to the pediatrics clinic for growth failure and developmental delay. Control DNA samples from Han Chinese individuals (n ¼ 209) were also genotyped. All samples were taken after informed consent was obtained, and blood samples were coded to maintain confidentiality. DNA isolation
The Biomed Blood Genomic DNA Purification Mini Kit (DN03) was used to isolate genomic DNA from the blood of the patient, the parents, and control samples. The extracted DNA samples were stored at 20 C until analysis. Polymerase chain reaction and DNA sequencing
Based on a candidate gene approach, all exons and the flanking intron-exon boundaries of CSA were amplified in the proband by polymerase chain reaction. All primers were synthesized according to
263
previous reports.8,9 The sequences were compared with the genomic sequences of CSA by BLAST search.
Results Clinical investigation
The patient is a 7-year-old boy with a 6-year history of growth failure and developmental delay, manifesting typical characteristics of Cockayne syndrome, such as a short stature of only 97 cm, a low weight of 12 kg, and the delay of photosensitivity developed at around 1 year of age. His skin became red after sun exposure with desquamation in a few days, leaving behind areas of gray pigmentation on the skin. He did not perspire even in the summer. His incisor and canine teeth were decayed, and he lost all his molars. He had a small head, gray and coarse hair, a distinctly dysmorphic face with sunken eyes, sharp nose and chin, and big ears. He had a progeroid appearance with loss of subcutaneous fat. He also had increased tone in the lower limbs with hyperreflexia and restriction of dorsiflexion at the ankles. Neurological examination revealed noticeable developmental delay, motor impairment, spastic paraparesis, and cerebellar ataxia. Both palms displayed single skin creases. Abdominal ultrasound and echocardiogram showed normal results. Magnetic resonance imaging of the brain revealed demyelination of the cerebral white matter, cortical atrophy, and ventricular dilatation. The cerebellum, brainstem, and corpus callosum were atrophic (Supplementary Fig S1A-D). In contrast, the CT scan result of his head at age 1 was normal (data not shown). Ocular
FIGURE 1. The sequence of two novel heterozygous mutations. (A) The patient and his mother had a novel heterozygous deletion mutation in exon 4 (c.394_398delTTACA). The sequence of his father was normal. (B) The patient and his father had a novel heterozygous A to C mutation in the 30 end of intron 6 (c.551-2A>C). The sequence of his mother was normal. (Color version of figure is available in the online edition.)
264
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
ultrasound revealed smaller than normal lengths of both axis oculi of 19 mm. Furthermore, the right vitreous showed opacities. Funduscopy was not performed because of patient incompliance. His right ear threshold was 50 dB and left ear threshold was 70 dB, which suggested moderate deafness. Psychometric testing revealed an intelligence quotient of 50, corresponding to moderate to severe mental retardation. Mutation analysis of CSA
Two heterozygous mutations (c.551-2A>C and c.394_398delTTACA) were detected in CSA; they were demonstrated to be disease-causing mutations, because they were not present in at least 418 normal chromosomes sequenced in this study. The Cockayne syndrome patient described here has a maternal five-nucleotide deletion mutation (c.394_ 398delTTACA; Fig 1A), which causes the shifting of the open reading frame and produces a 136eamino acid premature protein. The former 132 amino acids are identical to those of the normal protein, whereas the rest of the protein is mutated. The patient has another paternal mutation, which affected the conserved acceptor site (AG) of canonical GTAG rule, located in 30 end of intron 6 (c.5512A>C; Fig 1B). Discussion
The protein encoded by the CSA gene contains WD-40 repeats,2 a motif known to be involved in protein-protein interactions. Laugel et al.4 summarized all the reported mutations of CSA. All 8 reported distinct missense mutations were located in the conserved WD domain, which suggests that the integrity of WD domains played a key role in the biologic functions of CSA. 6 splicing mutations were reported, including a large deletion of exon 4 and inversion in intron 4 (r.[844_1122del, 1041_1122del], c.[275þ703_399þ347del; 399þ348_399þ2007inv; 399þ 2008 399þ2558delins8]; Fig 2). Furthermore, we also indicated the newly identified splicing mutation in this figure. Among them, reported C-terminal truncate mutation (e.g., p.Tyr322X)4 and N-terminal truncate mutation (e.g., p.Glu13X)9 seem to be equally capable of impairing their
biologic functions and inducing complete Cockayne syndrome symptoms. Notably, one case was reported without any detectable mutations in the canonic donor site and acceptor site; however, unusual splicing variants were found and no full-length messenger RNA (mRNA) in the patient,10 indicating that some other mutations affecting the alternative splicing may exist in the intron or other regulatory sequence. All in-frame deletions, either genomic deletions or splicing defects, affect at least one WD domain.4 In this study, we identified two novel mutations in the patient: a novel five-nucleotide frame-shifting deletion and a distinct splice mutation. The maternal five-nucleotide deletion mutation (c.394_398delTTACA) results in the shifting of the open reading frame and is likely to produce a 136eamino acid premature protein, in which the last 3 WD domains are lost. The paternal mutation is a novel splice acceptor site mutation, an A to C transversion at the 2 position of intervening sequence 6 (c.551-2A>C), which changed the splice acceptor site AG to CG, thus possibly causing alternative splicing.11 To analyze the outcome of the mutation, we collected peripheral blood samples from the patient’s father and extracted the total RNA. In fact, we only amplified the normal mRNA of CSA but failed to identify the abnormal transcript. As we know, the father carried both normal and mutated CSA alleles; we assumed that the mutated form of mRNA is more vulnerable to degradation and difficult to be detected. It is commonly known that splicing defects can lead to potentially harmful protein variants. mRNAs are subject to quality control in the nucleus, thus preventing the export of splicing-defective transcripts.12 Kleppa et al. reported a splice acceptor site mutation, a G to A transition in the 1 position of intervening sequence 6 (c.551-1G>A), which changed the splice acceptor site AG to AA.13 This mutation also affected the same splice site in our study. They found that the abnormal mRNA yielded a frameshift from codon 184 (G to D) and ended in a premature stop codon (X) at codon 210. This suggests that different mutations that affect the same splice site may cause different splicing. Recently, the study by Kamenisch et al.14 revealed that CSA and CSB proteins are not only present in the nucleus but also recruited to the mitochondrion upon oxidative stress. Furthermore, they directly interact with mitochondrial DNA and base excision repaireassociated 8-oxoguanine DNA glycosylase mitochondrial, as well as single-stranded DNA binding protein 1, mitochondrial. Their interactions protect against aging- and stress-induced mitochondrial DNA mutations and the apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models and human progeroid syndromes.14 This study links a novel role of intramitochondrial Cockayne syndrome proteins and suggests that more details need to be clarified for the functions of CSA. Conclusion
FIGURE 2. The schematic diagram shows the CSA splicing mutation reported. The red rectangle stands for the exon, the black for the intron, and the mutation with asterisk is the mutation identified in this article. (Color version of figure is available in the online edition.)
In summary, we report a new paternal splice acceptor site mutation (c.551-2A>C) and a novel maternal fivenucleotide deletion mutation (c.394_398delTTACA) found in a Chinese family. These mutations extend the mutation spectrum of Cockayne syndrome in the context of the Chinese race, further providing the possibility of prenatal
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
diagnosis for future offsprings in this family. In fact, we did provide the prenatal diagnosis for this family by taking the umbilical cord blood at 5 months of pregnancy; unfortunately, the fetus carried the same mutations reported here as his brother. This work was supported by the Leading Academic Discipline Project of the Beijing Education Bureau. The authors sincerely thank all of the patients who participated in this study.
Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.pediatrneurol.2015.06.006. References 1. Cockayne EA. Dwarfism with retinal atrophy and deafness. Arch Dis Child. 1936;11:1-8. 2. Henning KA, Li L, Iyer N, et al. The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell. 1995;82:555-564. 3. Troelstra C, Gool AV, Wit JD, Vermeulen W, Bootsma D, Hoeijmakers JH. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s syndrome and preferential repair of active genes. Cell. 1992;71:939-953. 4. Laugel V, Dalloz C, Durand M, et al. Mutation update for the CSB/ ERCC6 and CSA/ERCC8 genes involved in Cockayne syndrome. Hum Mutat. 2010;31:113-126.
265
5. Fousteri M, Vermeulen W, van Zeeland AA, Mullenders LH. Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol Cell. 2006;23:471-482. 6. Groisman R, Polanowska J, Kuraoka I, et al. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003; 113:357-367. 7. Kleijer WJ, Laugel V, Berneburg M, et al. Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Repair (Amst). 2008;7:744-750. 8. Rapin I, Lindenbaum Y, Dickson DW, Kraemer KH, Robbins JH. Cockayne syndrome and xeroderma pigmentosum: DNA repair disorders with overlaps and paradoxes. Neurology. 2000;55: 1442-1449. 9. Cao H, Williams C, Carter M, Hegele RA. CKN1 (MIM 216400): mutations in Cockayne syndrome type A and a new common polymorphism. J Hum Genet. 2004;49:61-63. 10. Komatsu A, Suzuki S, Inagaki T, Yamashita K, Hashizume K. A kindred with Cockayne syndrome caused by multiple splicing variants of the CSA gene. Am J Med Genet A. 2004;128A:67-71. 11. Fogel BL, Lee JY, Perlman S. Aberrant splicing of the senataxin gene in a patient with ataxia with oculomotor apraxia type 2. Cerebellum. 2009;8:448-453. 12. Hocine S, Singer RH, Grünwald D. RNA processing and export. Cold Spring Harb Perspect Biol. 2010;2:a000752. 13. Kleppa L, Kanavin ØJ, Klungland A, Strømme P. A novel splice site mutation in the Cockayne syndrome group A gene in two siblings with Cockayne syndrome. Neuroscience. 2007;145:1397-1406. 14. Kamenisch Y, Fousteri M, Knoch J, et al. Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging. J Exp Med. 2010; 207:379-390.
Contents lists available at ScienceDirect
Pediatric Neurology journal homepage: www.elsevier.com/locate/pnu
Clinical Observations
Two Novel Heterozygous Mutations in ERCC8 Cause Cockayne Syndrome in a Chinese Patient Yun-pu Cui MD a, Yi-yu Chen MMed b, c, Xue-mei Wang MD a, Xin-li Wang MD, PhD a, *, Xu Nan BS c, d, Hongshan Zhao PhD c, d, * a
Department of Pediatrics, Peking University Third Hospital, Beijing, China Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing, China c Human Disease Genomics Center, Peking University, Beijing, China d Department of Medical Genetics, School of Basic Medical Sciences, Peking University, Beijing, China b
abstract BACKGROUND: Cockayne syndrome (MIM #133540, Cockayne syndrome B; 216400, Cockayne syndrome A) is a rare
autosomal recessive inherited disease in which the characteristic symptoms are premature aging, cachectic dwarfism, lack of subcutaneous fat, neurological alterations, light sensitivity, and failure to thrive. The mutated gene responsible for this syndrome has been identified as usually either CSA (CKN1, ERCC8) or CSB (ERCC6). In this study, we describe the case of a 7-year-old Chinese boy with characteristic symptoms of Cockayne syndrome A and the conduction of mutation screening of the CSA gene. METHODS: The patient was diagnosed with Cockayne syndrome in the pediatrics clinic for growth failure and developmental delay. We collected peripheral blood samples of the patient and his parents and then extracted the genomic DNA. DNA samples from control subjects and the patient were subjected to polymerase chain reaction amplification. All exons and the flanking intron-exon boundaries of CSA were amplified; then, the polymerase chain reaction products were directly sequenced for mutation screening. RESULTS: Two novel heterozygous CSA mutations, c.551-2A>C and c.394_398delTTACA, were identified in the patient. The c.551-2A>C mutation originates from his father and changed the splice acceptor site AG to CG, thus possibly causing alternative splicing. The c.394_398delTTACA from his mother caused a frameshift after the amino acid at position 132, thus introducing a premature stop codon in the gene sequence. CONCLUSIONS: These mutations extend the mutation spectrum of Cockayne syndrome in the context of Chinese race and provide possibilities of prenatal diagnosis for future offsprings in this family. Keywords: Cockayne syndrome, CSA, autosomal recessive, mutation
Pediatr Neurol 2015; 53: 262-265 Ó 2015 Elsevier Inc. All rights reserved.
Cockayne syndrome (MIM #133540, 216400) is a rare autosomal recessive inherited disease in which the characteristic symptoms are premature aging, cachectic dwarfism, lack of subcutaneous fat, neurological alterations, light sensitivity, and failure to thrive. Since first described by Cockayne in 1936,1 about 150-200 cases have been reported worldwide, and the mutated gene responsible for Y.-p.C. and Y.-y.C. contributed equally to this work. Conflict of interest: Nothing to declare.
Article History: Received November 26, 2013; Accepted in final form March 15, 2014 * Communications should be addressed to: Dr. Wang; Department of Pediarics; Peking University Third Hospital; Dr. Zhao; Department of Medical Genetics; Peking University Health Science Center; Human Disease Genomics Center; Peking University; Beijing, China. E-mail addresses: [email protected], [email protected] 0887-8994/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pediatrneurol.2015.06.006
this syndrome has been identified as usually either CSA (CKN1, ERCC8)2 or CSB (ERCC6).3 Cockayne syndrome, xeroderma pigmentosum (MIM #278700, 278720, 278730, 278740, 278760, 278780, 610651), and trichothiodystrophy (MIM #601675) constitute the nucleotide excision repair (NER) disorder family.4 The role that CSA protein plays in NER is revealed: when the elongating RNAPIIo encounters DNA lesions, the RNA polymerase II (RNAPIIo) is stalled and this triggers the recruitment of the NER proteins; CSA is dispensable for the attraction of NER proteins, whereas cooperation with CSB is required to recruit XPA binding protein 2 (XAB2), high mobility group nucleosome binding domain 1 (HMGN1), and transcription elongation factor A (SII)(TFIIS); and CSB serves as a repair coupling factor to recruit other NER proteins, chromatin remodelers, and CSAE3-ubiquitin ligase complexes to the stalled RNAPIIo.5,6
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
It is reported that the incidence of Cockayne syndrome in western European is 2.7 per million.7 However, there were no incidences, and only one mutation report of CSB was found in the Chinese population until now. In this article, we report a new paternal splice acceptor site mutation (c.551-2A>C) and a maternal five-nucleotide deletion mutation (c.394_398delTTACA) found in a Chinese family. These mutations extend the mutation spectrum of Cockayne syndrome in the context of Chinese race and provide possibilities of prenatal diagnosis for future offsprings in this family. Materials and Methods Patient and control samples
The patient is a 7-year-old boy of Han nationality who was the firstborn child of nonconsanguineous parents. He was brought to the pediatrics clinic for growth failure and developmental delay. Control DNA samples from Han Chinese individuals (n ¼ 209) were also genotyped. All samples were taken after informed consent was obtained, and blood samples were coded to maintain confidentiality. DNA isolation
The Biomed Blood Genomic DNA Purification Mini Kit (DN03) was used to isolate genomic DNA from the blood of the patient, the parents, and control samples. The extracted DNA samples were stored at 20 C until analysis. Polymerase chain reaction and DNA sequencing
Based on a candidate gene approach, all exons and the flanking intron-exon boundaries of CSA were amplified in the proband by polymerase chain reaction. All primers were synthesized according to
263
previous reports.8,9 The sequences were compared with the genomic sequences of CSA by BLAST search.
Results Clinical investigation
The patient is a 7-year-old boy with a 6-year history of growth failure and developmental delay, manifesting typical characteristics of Cockayne syndrome, such as a short stature of only 97 cm, a low weight of 12 kg, and the delay of photosensitivity developed at around 1 year of age. His skin became red after sun exposure with desquamation in a few days, leaving behind areas of gray pigmentation on the skin. He did not perspire even in the summer. His incisor and canine teeth were decayed, and he lost all his molars. He had a small head, gray and coarse hair, a distinctly dysmorphic face with sunken eyes, sharp nose and chin, and big ears. He had a progeroid appearance with loss of subcutaneous fat. He also had increased tone in the lower limbs with hyperreflexia and restriction of dorsiflexion at the ankles. Neurological examination revealed noticeable developmental delay, motor impairment, spastic paraparesis, and cerebellar ataxia. Both palms displayed single skin creases. Abdominal ultrasound and echocardiogram showed normal results. Magnetic resonance imaging of the brain revealed demyelination of the cerebral white matter, cortical atrophy, and ventricular dilatation. The cerebellum, brainstem, and corpus callosum were atrophic (Supplementary Fig S1A-D). In contrast, the CT scan result of his head at age 1 was normal (data not shown). Ocular
FIGURE 1. The sequence of two novel heterozygous mutations. (A) The patient and his mother had a novel heterozygous deletion mutation in exon 4 (c.394_398delTTACA). The sequence of his father was normal. (B) The patient and his father had a novel heterozygous A to C mutation in the 30 end of intron 6 (c.551-2A>C). The sequence of his mother was normal. (Color version of figure is available in the online edition.)
264
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
ultrasound revealed smaller than normal lengths of both axis oculi of 19 mm. Furthermore, the right vitreous showed opacities. Funduscopy was not performed because of patient incompliance. His right ear threshold was 50 dB and left ear threshold was 70 dB, which suggested moderate deafness. Psychometric testing revealed an intelligence quotient of 50, corresponding to moderate to severe mental retardation. Mutation analysis of CSA
Two heterozygous mutations (c.551-2A>C and c.394_398delTTACA) were detected in CSA; they were demonstrated to be disease-causing mutations, because they were not present in at least 418 normal chromosomes sequenced in this study. The Cockayne syndrome patient described here has a maternal five-nucleotide deletion mutation (c.394_ 398delTTACA; Fig 1A), which causes the shifting of the open reading frame and produces a 136eamino acid premature protein. The former 132 amino acids are identical to those of the normal protein, whereas the rest of the protein is mutated. The patient has another paternal mutation, which affected the conserved acceptor site (AG) of canonical GTAG rule, located in 30 end of intron 6 (c.5512A>C; Fig 1B). Discussion
The protein encoded by the CSA gene contains WD-40 repeats,2 a motif known to be involved in protein-protein interactions. Laugel et al.4 summarized all the reported mutations of CSA. All 8 reported distinct missense mutations were located in the conserved WD domain, which suggests that the integrity of WD domains played a key role in the biologic functions of CSA. 6 splicing mutations were reported, including a large deletion of exon 4 and inversion in intron 4 (r.[844_1122del, 1041_1122del], c.[275þ703_399þ347del; 399þ348_399þ2007inv; 399þ 2008 399þ2558delins8]; Fig 2). Furthermore, we also indicated the newly identified splicing mutation in this figure. Among them, reported C-terminal truncate mutation (e.g., p.Tyr322X)4 and N-terminal truncate mutation (e.g., p.Glu13X)9 seem to be equally capable of impairing their
biologic functions and inducing complete Cockayne syndrome symptoms. Notably, one case was reported without any detectable mutations in the canonic donor site and acceptor site; however, unusual splicing variants were found and no full-length messenger RNA (mRNA) in the patient,10 indicating that some other mutations affecting the alternative splicing may exist in the intron or other regulatory sequence. All in-frame deletions, either genomic deletions or splicing defects, affect at least one WD domain.4 In this study, we identified two novel mutations in the patient: a novel five-nucleotide frame-shifting deletion and a distinct splice mutation. The maternal five-nucleotide deletion mutation (c.394_398delTTACA) results in the shifting of the open reading frame and is likely to produce a 136eamino acid premature protein, in which the last 3 WD domains are lost. The paternal mutation is a novel splice acceptor site mutation, an A to C transversion at the 2 position of intervening sequence 6 (c.551-2A>C), which changed the splice acceptor site AG to CG, thus possibly causing alternative splicing.11 To analyze the outcome of the mutation, we collected peripheral blood samples from the patient’s father and extracted the total RNA. In fact, we only amplified the normal mRNA of CSA but failed to identify the abnormal transcript. As we know, the father carried both normal and mutated CSA alleles; we assumed that the mutated form of mRNA is more vulnerable to degradation and difficult to be detected. It is commonly known that splicing defects can lead to potentially harmful protein variants. mRNAs are subject to quality control in the nucleus, thus preventing the export of splicing-defective transcripts.12 Kleppa et al. reported a splice acceptor site mutation, a G to A transition in the 1 position of intervening sequence 6 (c.551-1G>A), which changed the splice acceptor site AG to AA.13 This mutation also affected the same splice site in our study. They found that the abnormal mRNA yielded a frameshift from codon 184 (G to D) and ended in a premature stop codon (X) at codon 210. This suggests that different mutations that affect the same splice site may cause different splicing. Recently, the study by Kamenisch et al.14 revealed that CSA and CSB proteins are not only present in the nucleus but also recruited to the mitochondrion upon oxidative stress. Furthermore, they directly interact with mitochondrial DNA and base excision repaireassociated 8-oxoguanine DNA glycosylase mitochondrial, as well as single-stranded DNA binding protein 1, mitochondrial. Their interactions protect against aging- and stress-induced mitochondrial DNA mutations and the apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models and human progeroid syndromes.14 This study links a novel role of intramitochondrial Cockayne syndrome proteins and suggests that more details need to be clarified for the functions of CSA. Conclusion
FIGURE 2. The schematic diagram shows the CSA splicing mutation reported. The red rectangle stands for the exon, the black for the intron, and the mutation with asterisk is the mutation identified in this article. (Color version of figure is available in the online edition.)
In summary, we report a new paternal splice acceptor site mutation (c.551-2A>C) and a novel maternal fivenucleotide deletion mutation (c.394_398delTTACA) found in a Chinese family. These mutations extend the mutation spectrum of Cockayne syndrome in the context of the Chinese race, further providing the possibility of prenatal
Y.-p. Cui et al. / Pediatric Neurology 53 (2015) 262e265
diagnosis for future offsprings in this family. In fact, we did provide the prenatal diagnosis for this family by taking the umbilical cord blood at 5 months of pregnancy; unfortunately, the fetus carried the same mutations reported here as his brother. This work was supported by the Leading Academic Discipline Project of the Beijing Education Bureau. The authors sincerely thank all of the patients who participated in this study.
Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.pediatrneurol.2015.06.006. References 1. Cockayne EA. Dwarfism with retinal atrophy and deafness. Arch Dis Child. 1936;11:1-8. 2. Henning KA, Li L, Iyer N, et al. The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell. 1995;82:555-564. 3. Troelstra C, Gool AV, Wit JD, Vermeulen W, Bootsma D, Hoeijmakers JH. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s syndrome and preferential repair of active genes. Cell. 1992;71:939-953. 4. Laugel V, Dalloz C, Durand M, et al. Mutation update for the CSB/ ERCC6 and CSA/ERCC8 genes involved in Cockayne syndrome. Hum Mutat. 2010;31:113-126.
265
5. Fousteri M, Vermeulen W, van Zeeland AA, Mullenders LH. Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol Cell. 2006;23:471-482. 6. Groisman R, Polanowska J, Kuraoka I, et al. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003; 113:357-367. 7. Kleijer WJ, Laugel V, Berneburg M, et al. Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Repair (Amst). 2008;7:744-750. 8. Rapin I, Lindenbaum Y, Dickson DW, Kraemer KH, Robbins JH. Cockayne syndrome and xeroderma pigmentosum: DNA repair disorders with overlaps and paradoxes. Neurology. 2000;55: 1442-1449. 9. Cao H, Williams C, Carter M, Hegele RA. CKN1 (MIM 216400): mutations in Cockayne syndrome type A and a new common polymorphism. J Hum Genet. 2004;49:61-63. 10. Komatsu A, Suzuki S, Inagaki T, Yamashita K, Hashizume K. A kindred with Cockayne syndrome caused by multiple splicing variants of the CSA gene. Am J Med Genet A. 2004;128A:67-71. 11. Fogel BL, Lee JY, Perlman S. Aberrant splicing of the senataxin gene in a patient with ataxia with oculomotor apraxia type 2. Cerebellum. 2009;8:448-453. 12. Hocine S, Singer RH, Grünwald D. RNA processing and export. Cold Spring Harb Perspect Biol. 2010;2:a000752. 13. Kleppa L, Kanavin ØJ, Klungland A, Strømme P. A novel splice site mutation in the Cockayne syndrome group A gene in two siblings with Cockayne syndrome. Neuroscience. 2007;145:1397-1406. 14. Kamenisch Y, Fousteri M, Knoch J, et al. Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging. J Exp Med. 2010; 207:379-390.
