Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant

Accepted Manuscript Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant

Yajuan Gu, Xiaodan Chang, Shan Dai, Qinghua Song, Hongshan Zhao, Pengcheng Lei PII: DOI: Reference:

S0378-1119(17)30507-3 doi: 10.1016/j.gene.2017.06.057 GENE 42020

To appear in:

Gene

Received date: Revised date: Accepted date:

6 April 2017 24 June 2017 28 June 2017

Please cite this article as: Yajuan Gu, Xiaodan Chang, Shan Dai, Qinghua Song, Hongshan Zhao, Pengcheng Lei , Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant, Gene (2017), doi: 10.1016/j.gene.2017.06.057

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Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant Yajuan Gu1*, Xiaodan Chang2*, Shan Dai2, Qinghua Song2#, Hongshan Zhao1,3#, Pengcheng

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Lei2

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1 Department of Medical Genetics, School of Basic Medical Sciences, Peking University,

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Beijing, China

2 Department of Dermatology, Peking University Third Hospital, Beijing, China

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3 Human Disease Genomics Center, Peking University, Beijing, China

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Correspondence to: H.-s. Zhao, Department of Medical Genetics, Human Disease Genomics Center, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Haidian

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District, Beijing 100191, China. Tel.: +86 10 82802846 ext 420; fax: +86 10 82801149.

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Correspondence to: Q.-h. Song, Department of Dermatology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China. Tel.: +86

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13661306467; fax: +86 10 62081686.

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E-mail addresses: [email protected] (H. Zhao) , [email protected] (Q. Song). These authors contributed equally to this work.

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Abstract Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. We

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collected two unrelated Chinese patients showing typical symptoms of XPC without

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neurologic symptoms. Direct sequencing of XPC gene revealed that patient 1 carried

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IVS1+1G>A and c.958 C>T mutations, and patient 2 carried c.545_546delTA and c.2257_2258insC mutations. All these four mutations introduced premature terminal codons

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(PTCs) in XPC gene. The nonsense mutation c.958 C>T yielded truncated mutant Q320X,

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and we studied its function for global genome repair kinetics. Overexpressed Q320X mutant can localize to site of DNA damage, but it is defective in CPD and 6-4PP repair. Readthrough

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of PTCs is a new approach to treatment of genetic diseases. We found that aminoglycosides

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could significantly increase the full length protein expression of Q320X mutant, but NER

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defects were not rescued in vitro.

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Key words Xeroderma pigmentosum, complementation group C (XPC), Mutation, Nucleotide excision repair (NER),

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Premature termination codons,

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Readthrough.

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Introduction Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. Patients may also have mental retardation and neurologic symptom (1). Early studies

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indicated the existence of eight genetic complementation groups designated XPA-XPG as

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well as the variant form XPV. At the same time, the oncogenes are named XPA to XPG and

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XPV. Classical XP patients have a defect in one of the seven XP genes, XPA to XPG, which belong to the nucleotide excision repair (NER) pathway (2, 3). Nucleotide excision repair

the

UV-induced

cyclobutane

pyrimidine

dimer

(CPD)

and

the

(6-4)

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including

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(NER) is a major pathway for removing a wide variety of helix-distorting DNA base lesions,

pyrimidine-pyrimidone photoproduct (6-4PP). Transcription-coupled repair (TCR) and global

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genome repair (GCR) are two sub-pathways of NER. There are 55 mutations predicted to be

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highly intolerant in eight genes related to XP namely DDB2, ERCC2, ERCC3, ERCC4, ERCC5, POLH, XPA and XPC (4).

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Xeroderma pigmentosum complementation group C (XPC, MIM 278720) is the most frequent

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complementation group worldwide and especially in USA, Europe, and the Middle East (5). Different with other complementation groups, XPC has defect in GCR while TCR function as normal. XPC gene act as a DNA damage sensor in GCR sub-pathway, in the form of heterotrimer consists of XPC, RAD23B and centrin2 and plays an essential role in initiation of the repair reaction (6, 7). Readthrough of premature terminal codons (PTCs) is a new approach to treatment of genetic disease, mainly targeting nonsense mutations promoted premature translational termination.

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cysticfibrosis (CF) and Duchenne muscular dystrophy were typical in this field, aminoglycosides were used to readthrough in vivo and partial correction of protein function in clinical trials(8). Nonsense mutations cause about 25% of the individual cases of XPC, based on HGMD database (http://www.hgmd.cf.ac.uk/ac/index.php). Kuschal et al. had extended

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research on Readthrough of XP PTC by aminoglycosides, resulting in increased expression of

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XPC protein and increased repair of 6-4PP and CPD (9).

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In this study, we described two unrelated Chinese patients showing typical symptoms of XP without neurologic symptoms and carried on direct sequencing of XPC gene. We identified

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four novel mutations in patients while not detected in unrelated normal controls. We also

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treated cells, which were transfected by c.958 C>T mutant plasmids, to observe the readthrough effects with aminoglycoside drugs. The results are helpful to further reveal the

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mechanisms and provide new clue for individual therapy of XPC.

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Materials and methods 1.1 Patient and Control Samples 1.1.1 Case 1 A 35-year-old Chinese female was referred to the dermatology clinic with chief complaints of

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hyperpigmented macules all over the body as well as photo-sensitivity and ulceration of a black

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skin rash on her left wing of nose for 18 months, with pain and itch. The history of development

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of pigmentation was revealed when she was a child. Blood sample from one of her normal

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sister was also collected. 1.1.2 Case 2

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A 18 year old male came with history of multiple hyperpigmented and hypopigmented macule all over his body. The above symptom was revealed when he was 7 months old. At the age of 8

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he developed a tumor on his nose which was removed by surgical operation and finally

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diagnosed as squamous cell carcinoma. His mother’s blood sample was also collected. The patient and controls gave their written, informed consent. The present study was

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approved by the Ethics Committee of the Peking University Health Science Center.

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Control DNA samples from Han Chinese individuals (n=50) were also genotyped.

1.2 Isolation of genomic DNA Peripheral blood samples were collected from Peking University Third Hospital. Genomic DNA was extracted using a Blood DNA Mini Kit (Biomed Biotechnologies, Inc., Beijing, PRC) according to the manufacturer’s instructions. DNA integrity and quantity were verified by agarose gel electrophoresis.

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1.3 Identification of XPC mutation DNA samples were subjected to PCR amplification. And we screened for XPC mutation by direct sequencing of PCR products from genomic DNA. Sixteen pairs of primers were used to amplify all the sixteen exons of XPC. Exon9 and exon16 were designed to be divided into two

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exon15 were amplified together. Primers are listed in the table I.

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fragments because they are too long and difficult for sequencing. Exon12, exon13 and exon14,

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After heating at 94°C for 5 minutes, polymerase chain reaction (PCR) amplification was performed with 35 cycles: 94°C for 30 seconds, annealing for 30 seconds(annealing

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temperatures are listed in table 1), 72°C for 30 seconds and followed by a final extension step

Biotechnologies, Inc., Beijing, PRC).

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at 72°C for 7 minutes. Then fragments were obtained for accurate sequencing (By AuGCT

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1.4 T-A cloning to confirm the mutation

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To determine the exact status of mutation, patient’s genomic DNA subjected to a second, independent amplification, using the same procedure as described above. PCR amplification

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produced a 792-base pair fragment was cloned into the pGEM®-T Easy Vectors (TIANGEN

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Biotech (Beijing) CO.LTD.) according to the manufacturer’s instructions. PCR products were inserted into vectors and sequenced in the By AuGCT Biotechnologies (Inc., Beijing, PRC) after 24h of amplification in TOP10 competent Escherichia coli cells (TIANGEN BIOTECH (Beijing) Co., Ltd, China) by heat shock at 42°C, according to manufacturer’s indications.

1.5 Construction of mutated XPC gene expression vector A full-length fragment of XPC was cloned by PCR from a human cDNA library. Constructs

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were cloned into the p3XFlag-CMV series of mammalian expression plasmids and verified by sequencing. XPC nonsense mutations (c.958 C>T) were constructed from the p3XFlag-CMV XPC plasmid using polymerase chain reaction-based mutagenesis strategy with specific primers as follows: forward (5’- CTGGTATTGTCTCTATAGCCAATTCCTCTGA-3’) and

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reverse (5’- TCAGAGGAATTGGCTATAGAGACAATACCAG -3’). After heating at 98°C

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for 2 minutes, polymerase chain reaction (PCR) amplification was performed with 17 cycles:

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98°C for 10 seconds, annealing 58°C for 30 seconds, 72°C for 10 minutes and followed by a final extension step at 72°C for 10 minutes. Then fragments were treated by Dpn I enzyme at

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37°C for 3 hours. After purification by TIANgel Midi Purification Kit (TIANGEN BIOTECH

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(Beijing) Co., Ltd, China) and 24h of amplification in TOP10 competent Escherichia coli cells (TIANGEN BIOTECH (Beijing) Co., Ltd, China) by heat shock at 42°C, according to

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manufacturer’s indications. The XPC nonsense mutations were verified by sequencing, named

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p3XFlag-CMV XPC-Q320X (Q320X).

1.6 Cell culture, transfections and treatments

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The Hacat cell line (kindly provided by Dr. Xiaoyan Qiu, Peking University, China) was

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maintained in Minimum Essential Medium (MEM) (ThermoFisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS). Logarithmically growing cells were transfected using NeofectTM DNA transfection reagent (Lingkechuangzhi, Beijing, China), according to the manufacturer’s instructions. Readthrough effect was studied by adding G418 (Dose of G418 is 100/200/400 μg/ml), one of aminoglycosides into the culture medium for 24 hours. The readthrough effect experiment was repeated twice.

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1.7 Antibodies Anti-XPC antibody was purchased from ABclonal Biotech Co., Ltd (ABclonal Biotech Co., Ltd, China). Anti-FLAG antibody was purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-6-4PP (64M-2) antibody and anti-CPD antibody were purchased from Santa

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Cruz Biotechnology (Cosmo Bio Co., Ltd), and MBL International (Woburn, MA),

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respectively, kindly provided by Dr. Jianyuan Luo.

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1.8 Preparation of cell extracts and western blot

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All samples were prepared in radio immune precipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and heated to 95°C for 10 min before separation and transfer of proteins to

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nitrocellulose (NC) membranes. Membranes were blocked in 5%(w/v) bovine serum albumin (BSA) powder/Tris-buffered saline with 005% (v/v) Tween (TBS-T) and then probed using

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the primary antibody in 5% (w/v) BSA blocking buffer at 4°C overnight. The membranes were then incubated with Alexa Fluor 680 (Rockland, Philadelphia, PA, USA) or IRDye 800 (Rockland)-labelled secondary antibodies at room temperature for 2 h. After three washes, the

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blots were developed using an Odyssey Infrared Imaging System (LI-COR Bioscience,

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Lincoln, NE, USA).

1.9 Local UV irradiation and immunofluorescence analyses Culture the cells in 35-mm glass-bottom dishes for one day after over expressed p3XFlag-CMV XPC and the mutant XPC (p3XFlag-CMV XPC-Q320X).

Wash cells twice

with ice-cold PBS and irradiate cells with UV (20 J/m2 of 254 nm UV for whole cell irradiation). Cells were then cultured for 20 minutes or 7 hours. Cells were fixed subsequently

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with 4% paraformaldehyde for 20 minutes and permeabilized with 0.2% TritonX-100 for 20 minutes. Pour 2 mL of 2M HCl and denature cellular DNA for 30 minutes at room temperature. The cells were then blocked with 10% FBS (v/v) for1 h at room temperature and exposed to anti-CPD antibody at 37°C for 30 minutes. After five washes with PBS, the cells

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were incubated with TRITC-conjugated secondary antibody. The cells were then exposed to

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anti-FLAG antibody at room temperature for 2 hours. After five washes with PBS, the cells

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were incubated with FITC-conjugated secondary antibody. Nuclei were stained with Hoechst 33342(H33342). The morphological features of the cells were observed and documented with

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an Olympus BX53F microscope (Olympus, Tokyo, Japan).

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1.10 RNA analyses

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific Inc., New York, NY,

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USA). cDNA was prepared by reverse transcription(RT) of the total RNA using the 5*All In One RT Master mix cDNA synthesis kit (Abcam, Cambridge, UK), according to the manufacturer’s protocol. The XPC mRNA level was assessed by PCR. The annealing

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temperature for the PCR was 58°C, and the programe included 24 cycles for XPC and 23

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cycles for GAPDH (internal control).The primer sequences were as follows: XPC, forward GTCCGAGATGTCACACAGAG, reverse AGTTCCCTAGAGCCCGCTTC; and GAPDH, forward CAAGGTCATCCATGAC AACTTTG, reverse GTCCACCACCCTGTTGCTGTAG. PCR products were separated by 2% agarose gel electrophoresisand stained with ethidium bromide (EB). Fig.4B is the statistics of two independent experiments.

1.11 Measurement of in vivo repair rates of UV-induced photolesions

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Transfect p3XFlag-CMV XPC and the mutant XPC (p3XFlag-CMV XPC-Q320X) into Hacat cells. Culture the cells in 35-mm glass-bottom dishes for 24 hours, treat the cells with or without 100μg/ml G418 after over expressed. After another 24 hours, cells were maintained at 37◦C for 2 h in medium containing 6mM thymidine to prevent dilution of DNA lesions by

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replication (11). Wash cells twice with ice-cold PBS and irradiate cells with UV (15 J/m2 of

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254 nm UV for whole cell irradiation). Cells were then cultured for the indicated times to

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allow DNA repair (0/1/3/7 hours). Genomic DNA was purified with the QIAamp DNA Blood Mini Kit (Qiagen), and the levels of remaining photolesions were determined using an

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enzyme linked immune sorbent assay with the lesion-specific antibodies 64M-2 according to

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the manufacturer’s instructions. There were three replicates of treatment.

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Results 2.1 Case report 2.1.1 The clinical feature of patient 1. Patient 1 was a 35-year-old female. When she was born, the whole body skin was red and some

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red macules were found on her face. As the age increased, the exposure area of skin gradually

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appeared freckle-like rashes which became more and spreading to the whole body. The above

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symptom aggravated after exposure to sunlight. A black papule appeared on the left wing of

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nose 18 months ago (The patient asked for medical help on November 12, 2013), with progressive increase in the size of the lesion and ulceration. Special examine: The whole body

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skin is dry, and multiple different size of skin pigmentation wide spread on her face, trunk and upper limbs, with smooth surface and fusion in some areas. The patient had some black patches

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with peanut to bean size on the partes zygomatica and perinasal areas. We observed a black papule on the left wing of nose, which has a central ulcer with tan scab on the surface. The pathology of the lesion on the left wing of nose showed diffuse melanocytes infiltration can be

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seen in the lower epidermis to the whole dermis layer (Fig. 1B), with a large number of pigment

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granules, various cell size, dense chromatin. Suspicious melanocytes are visible in part of the small blood vessels. The lesion was positive for malignant melanoma. A positive family history and consanguinity was confirmed (Fig. 1A). 2.1.2 The clinical feature of patient 2. Patient 2 was a 18-year-old Chinese male. Her history revealed development of pigmentation at the age of 7 months old, especially in the area of sunlight exposed. With the increase of age, the lesions gradually spread to trunk and proximal limbs. A red papula on his nose was removed by

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operation and pathological diagnosis (from Xi Jing Hospital) showed it is squamous cell carcinoma after replase. Special examine: the patient has multiple, small, dark brown colored lentigines appeared as tanned pigmented macule and hypopigmented macule on the face, trunk and limbs, varying in size and shape. Postoperative scar can be seen on his nose. There was no

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positive history in his family (Fig. 2A).

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2.2 Mutation Analysis of XPC

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After direct sequencing of PCR products in two controls and patient 1, two novel

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heterozygous XPC mutations in the first exon-intron boundary and the eighth exon of were identified (Figure. 1). As shown, patient 1 (Fig. 1C) has splicing mutation (IVS1+1G>A) at

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the first exon-intron boundary (marked by the black frame). Patient1 and her sister carried a nonsence mutation c.958 C>T, which yields a premature mutant Q320X, in exon 8, while

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unrelated control individuals (Fig. 1D) are normal. Patient 2 and his mother (Fig. 2B) have a 2bp deletion mutation (c.545_546delTA) in exon5, which can yield a frame-shift from the 182 Aa and produce a premature 186Aa

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protein(p.Ile182LysfsX5). Another mutation in exon 13 was identified from patient 2 (Fig.

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2C). To clarify the mutation, T-A cloning was performed. Compared with wild-type (Fig.2E), the wild sequence is inserted by one base C (c.2257_2258insC) (Fig. 2D), which introduces the frame shift and generates a premature protein (p.Arg753ProfsX46).

2.3 The c.958C>A substitution results the truncated Q320X XPC Patient 1 carried a C to T nonsense mutation at base pair 958 that results in conversion of the glutamine codon at amino acid 320 to a stop codon. This would result in marked truncation of

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the XPC protein at amino acid 320 rather than at its full length of 940 amino acids (Molecular weight 37kDa). Cells transfected with Q320X mutants express a truncated XPC protein, its molecular weight was consistent with expected (Fig. 3C), about 40 kDa. Bioinformatics analysis suggests that the mutated glutamine of XPC is highly conserved throughout species

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(Fig. 3A) and the C terminal of XPC interacting with RAD23 and CETN2 are missing after

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the substitute. The functional domain which recognizes damaged DNA doesn’t expression

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any more either (Fig. 3B). The database Genecards shows that the 390-395 amino acids are the nuclear localization signal. In order to confirm the mutation’s effect on sub-cellular

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localization and the ability of recognizing CPD damage, immunofluorescence analyses were

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performed. The location of Q320X mutant showed similar pattern as wild typein Hacat cell line (shown as ND, non-damaged), and it could recognize CPD 20 minutes later after UV

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irradiation. The consequence of 7 hours later after irradiation showed that the ability of

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repairing CPD was decreased in Q320X (Fig. 3D).

2.4 The Q320X mutant abolished GGR function in vivo.

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In order to investigated whether the Q320X mutant can physical function in GGR in vivo, the

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ability of removing 6-4PP was measured. To avoid the influence of endogenous XPC, we designed siRNAs against 3’UTR region of XPC mRNA and transfected them to Hacat cells respectively. It was proved that si3177 can knockdown XPC either in the form of mRNA or protein (Fig. 4B, C). 3XFLAG-XPC (wild type or Q320X mutant) were transfected into Hacat cells after endogenous XPC were knockdown by si3177. Cells were irradiated at 15 J/m2 and were harvested at various time points thereafter. The remaining 6-4PPs were measured using lesion-specific monoclonal antibodies. It showed that Q320X mutant had no difference in the

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remaining of 6-4PP with endogenous knockdown MOCK, while wild type XPC removing lesion completely comparing with MOCK (showed in Fig. 4D).

2.5 G418 can readthrough full length XPC in vivo but GGR defect was not rescued by it. Readthrough of premature terminal codons (PTCs) is a new approach to treatment of genetic

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disease, mainly targeting nonsense mutations promoted premature translational termination.

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Western blot was performed to measure the levels of XPC protein in transfected Hacat cells

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with or without G418 treatment (also known as Geneticin). 24h incubation with G418 led to

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20–60% increase of full length XPC protein under different concentrations. (Fig. 4A). As described previously, the remaining 6-4PPs were measured using a lesion-specific monoclonal

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antibody after incubated with G418. At 7 h after UV exposure, cells transfected with Q320X mutant had about 40% of the 6–4PPs remaining after G418 treatment, which didn’t show

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obvious difference with treatment free group (Fig. 4D). It suggested that the readthrough

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induced by G418 couldn’t improve the abilities for DNA damage removal.

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Discussion Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. Patients may also have mental retardation and neurologic symptom (1). Xeroderma

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pigmentosum complementation group C (XPC, MIM 278720) is the most frequent

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complementation group worldwide and especially in USA, Europe, and the Middle East (4).

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Here we identified IVS1+1G>A and c.958 C>T mutations responsible for a Chinese patient

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suffered from Xeroderma pigmentosum complementation group C, and c.545_546delTA and c.2257_2258insC mutations responsible for the another patient. These four mutations were

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not collected in HGMD database (http://www.hgmd.cf.ac.uk/ac/index.php) and Ensembl database （http://asia.ensembl.org/index.html?redirect=no）, which indicated that all these

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mutation probably are first reported in the world.

Through mutation analysis, the mutated allele was found to have a splicing mutation,

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IVS1+1G>A, that breaks the rule of GT-AG. The G > A substitution at the splicing donor site of intron 1 alters the obligatory GT donor dinucleotide to AG or AT, probably introduces

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alternatively spliced XPC mRNA and generates abnormal molecules (12). The nonsense mutation c.958 C>T converts the CAG codon of glutamine at amino acid 320 to a UAG stop codon resulting in marked truncation of the 940 amino acid XPC protein, while the NER function motif are lost(Fig. 3B).

Deletion or insertion mutations often result frameshift. We identified c.545_546delTA and c.2257_2258insC in patient 2, c.545_546delTA could yield a frame-shift from the 182 amino

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acid, which is predicted to result in a truncated protein absent of function domain for NER initiation. The insertion mutation c.2257_2258insC alters the XPC reading frame and results in the introduction of a stop codon after 753 residues, which is predicted to result in a truncated, protein absent of predicted interacting with TFIIH and CETN 2(Fig. 3B).

database

Genecards

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The

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(http://www.genecards.org/cgi-bin/carddisp.pl?gene=XPC&search=dac294fa08da465d37382

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55fc128ff20) shows that the 390-395 amino acids are the nuclear localization signal. Q320X

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mutants express a truncated XPC protein absent of potential nuclear localization signals (NLS), we suspected that it couldn’t locate in nuclear any more. However,

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immunofluorescence experiments showed the mutant had the same location as wild type (Fig. 3D, shown as ND, non-damaged). We suspected that XPC has some other NLS. Analysis the of

XPC

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sequence

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protein

in

prediction

website

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http://www.sbc.su.se/~maccallr/nucpred/ http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi52-57

NLS, and

confirmed

our

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assumption, showing that the region of 52-57 amino acids also is the potential nuclear

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localization signal.

Recently，researchers are interest in therapeutic approaches aimed at readthrough of in-frame PTCs to enable synthesis of full-length proteins. We tried to use aminoglycoside drugs to recover the full length expression of Q320X mutant and rescue its function. Our results showed that synthesis of full-length XPC protein can recover to 69 percent by readthrough of PTCs using G418, while NER defects were not rescued. We speculate that better cell model should be set up, such as cells obtained from XPC patients or other XPC defective cells (13,

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14). The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. To establish the gene knockout or mutation knockin cell lines by genome editing technology will benefit future studies (15).

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The efficiency of aminoglycoside-mediated readthrough depends on the type and copy

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number of PTC (UGA>UAG>UAA) (16-19), the downstream 4+nucleotide（C>U>A>G）(20).

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In this study, the c.958 C>T substitution introduce UAG into XPC, the downstream

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4+nucleotide is C, thus we predicted that it can be readthrough in a high efficiency. In addition, it was proved that the stop codons UAG and UAA direct insertion of glutamine in

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prokaryotes (21), while the ancestor of the PTC is glutamine. We hope to provide some new clue for the therapy of patient 1. However, unlike what we expected, NER defect of Q320X

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was not rescued. Our results suggested that the mechanisms about PTCs and functional recovery still need to explore. Since systemic aminoglycoside administration may be associated with severe side effects such as kidney damage and hearing loss, and G418 showed

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greater toxicity compared to all other compounds in published article (22), a series of

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small-molecule nonaminoglycoside compounds were obtained by high throughput screening assay, the most famous one is PTC124 (23). Treatment with PTC124, BZ16, and RTC14 (small-molecule nonaminoglycoside compounds) resulted in similarly increased XPC mRNA expression and photoproduct removal with less toxicity than with the aminoglycosides (10). Since then, we will try to use small-molecule nonaminoglycoside compounds, such as PTC124, for observing the readthrough effects of Q320X mutant.

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In summary, we identified four novel mutations from two unrelated XPC patients. Furthermore, we carried out the function study of Q320X mutant try to recover its biology activities by PTC. Our results will provide the basis for further genetic counseling and prenatal gene diagnosis in these families and contribute to the individual therapy strategy of

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Funding This work was supported by the Leading Academic Discipline Project of Beijing Education

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Bureau.

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Acknowledgments

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We gratefully acknowledge the patients for having participated in this study.

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Politano L, Nigro G, Nigro V, Piluso G, Papparella S, Paciello O, Comi LI. 2003, Gentamicin administration in Duchenne patients with premature stop codon.

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Preliminary results. Acta Myol, 22, 15-21. 9.

Kuschal C, DiGiovanna JJ, Khan SG, Gatti RA, Kraemer KH. 2013, Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons.Proc Natl Acad Sci U S A, 110, 19483-8. Yasuda G, Nishi R, Watanabe E, Mori T, Iwai S, Orioli D, Stefanini M, Hanaoka F,

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Sugasawa K. 2007, In vivo destabilization and functional defects of the xeroderma

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pigmentosum C protein caused by a pathogenic missense mutation. Mol Cell Biol, 27, 6606-14.

Bunick CG, Miller MR, Fuller BE, Fanning E, Chazin WJ. 2006, Biochemical and

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structural domain analysis of xeroderma pigmentosum complementation group C protein. Biochemistry, 45, 14965-79.

Cartault F, Nava C, Malbrunot AC, Munier P, Hebert JC, N'guyen P, Djeridi N,

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Pariaud P, Pariaud J, Dupuy A, Austerlitz F, Sarasin A. 2011, A new XPC gene splicing mutation has lead to the highest worldwide prevalence of xeroderma

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pigmentosum in black Mahori patients. DNA Repair (Amst), 10, 577-85.

Miller M, Chinsomboon N, Stefanini M, Kraemer KH. 2001, A stop codon in xeroderma pigmentosum group C families in Turkey and Italy: molecular genetic evidence for a common ancestor. J Invest Dermatol, 117, 197-204. 14.

Wang G, Chuang L, Zhang X, Colton S, Dombkowski A, Reiners J, Diakiw A, Xu XS.. 2004,

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role of XPC

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cisplatin DNA damaging

treatment-mediated cell cycle regulation. Nucleic Acids Res, 32, 2231-40.

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Wassef M, Luscan A, Battistella A, Le Corre S, Li H, Wallace MR, Vidaud M, Margueron R. 2017, Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines. Methods. 2017 May 10. pii: S1046-2023(16)30269-9. doi: 10.1016/j.ymeth.2017.05.003. [Epub ahead of print] Zilberberg A, Lahav L, Rosin-Arbesfeld R. 2010, Restoration of APC gene function in

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the adenomatous polyposis coli gene restores its biological activity in human cancer

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cells. PLoS One，6，e24125.

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Floquet C, Hatin I, Rousset JP, Bidou L. 2012, Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin.PLoS Genet, 8, e1002608.

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Nilsson M, Ryden-Aulin M. 2003, Glutamine is incorporated at the nonsense codons

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UAG and UAA in a suppressor-free Escherichia coli strain. Biochim Biophys Acta, 1627, 1-6. 22.

Kuschal C, Khan SG, Enk B, DiGiovanna JJ, Kraemer KH. 2015, Readthrough of stop codons by use of aminoglycosides in cells from xeroderma pigmentosum group C

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patients. Exp Dermatol, 24, 296-7.

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Figures

Figure 1. Pedigree and identification of mutations in the XPC gene of patient 1. A: Pedigree

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of patient 1, B: HE staining of the affected tissue. C: Patient 1 had a novel heterozygous

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splicing site mutation IVS1+1G>A and D: c.958 C>T (Q320X) nonsence mutation in exon8.

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Figure 2. Pedigree and identification of mutations in the XPC gene of patient 2. A: Pedigree

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of patient 2. B: Patient 2 carried heterozygous mutations c.545_546delTA in exon5. C, D, and

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E: Direct sequencing and T-A cloning showed that patient 2 carried c.2257_2258insC in

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exon13.

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Figure 3. The c.958C>A results the truncated Q320X XPC. A: Analyze the conservation of glutamine between species, including Macaca mulatta (Rhesus monkey), Bos taurus (cattle),

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Canis lupus familiaris (dog), Homo sapiens (human), Mus musculus (house mouse), Pan troglodytes (chimpanzee), Rattus norvegicus (Norway rat). B: Distribution of mutation in XPC. Position of mutation in XPC site with respect to known domains of human XPC,

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several previously determined interacting domains are indicated by color lines (11). Q320X

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lost domains for NER function. C: The Q320X mutant codes a truncated XPC protein, which molecular weight was consistent with expected. D: Hacat cells overexpressed with XPC (WT or G320X) were irradiated with UV 20 J/m2. After 20 minutes or 7 hours at 37 ◦C, the locations of XPC (green) and CPD (red) were visualized by immunofluorescence(40x). Nuclei were stained with Hoechst 33342(H33342).

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Figure 4. The Q320X mutant lost GGR function in vivo. G418 could improve the amount of full length XPC but GGR defects were not rescued. A: Increased XPC protein with G418.

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HaCat cells transfected with wild type or Q320X mutant plasmids were incubated with different concentrations of G418 for 24h and protein were measured. Normalized level of XPC protein in cells transfected wild type was defined as 100 percent.

Rescue efficiency

was calculated within group. Data are mean±SD of two independent experiments. B, C:

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si3177 can knockdown XPC either on mRNA and protein levels. D: Effect of Q320X mutant

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in removal of 6-4PPs with or without G418 treatment. MOCK was used as a negative control

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(transfected with 3XFLAG vector after endogenous XPC were knockdown by si3177).

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Tables Table I: Primers used for PCR amplification of XPC Product Primer Sense

Primer Anti-sense

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Exons

size (bp)

GCCTAGTACAAGAAGCTCCT

TTGGATCGGGCGAAGCTCG

684

2

TCTGTAGTCATAGGAGAAGTAA

GCATGCTGTCACTGTTCTTA

620

3

CAGCACATCTGGTAGTAATTAG

GGAAGAGAGTAAACAGCCTTC

540

4

ATAACACTCAGCACACTGCC

5

GTTTGCTGGTGAGAAGGAGC

6

GAAGGAATGAGTATACAGAGAG

7

TGGGTCTGCAGTGATGATAG

8

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480

CACCCTCCAAGACAGACACT

455

CTCTACCAGGGCAAGATCAT

532

GTCAACCATCTTTTTCTCCAG

934

TGATGAGCATGTTCCTGTTC

AAGTTCAGGCTGCTAGATAC

483

9a

CATACAACCCTGAAGGATAGC

ACACCTCTAGCCACTGGTCT

779

9b

TGGCCTCCAGGGTGTCTTAT

CGCGGCAGTTCATCTTTCAA

789

10

CTTCCTTGCAGACCTTTAAC

CACACGCACACTGGCTCA

599

ACAGGCAGTCCACGTTCAAG

ACAGGAGTCTGAGGCCTACT

525

12and13

GCCAAGATTGCACCACTGTA

AGTTTCATTGGCTCCGTTCC

792

14and15

AGCTTCCACAGGCCTGCCTT

GGACACTGGGACAGGGCTTG

1114

16a

TGCACTCAGCTCGTAGCAG

GGCTTCTGTCACCACAAAAT

1055

16b

TGCCTCCTAGACCTGTTCTT

ACCTGGGTGTTATTACCGAC

743

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AGGTGTCTCATTCAAGTCTCT

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Anneal temperature of exon 5, 8, 10 were 58°C, all the other were 56 °C.

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Abbreviations list (6-4) pyrimidine-pyrimidone photoproduct

CCK-8

Cell Counting Kit-8

CF

cystic fibrosis

CPD

cyclobutane pyrimidine dimer

CS

Cockayne syndrome

DMD

Duchenne muscular dystrophy

ELISA

enzyme-linked immunosorbent assay

GCR

global genomic repair

HRP

horseradish peroxidase

JNK

c-Jun N-terminal kinase pathway

NER

Nucleotide excision repair

NLS

nuclear localization signal

PCR

polymerase chain reaction

Q320X RISC

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Premature termination codons

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PTCs

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6-4PP

pCMV-FLAG-XPC-Q320X RNA inducedsilencing complex

RNAi

RNA interference

RT-PCR

reverse transcription-polymerase chain reaction

SNPs

Single nucleotide polymorphism

TCR

transcription-coupled repair

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TRAF3 interacting protein 3

TTD

trichothiodystrophy

W690S

XPC p.Trp690Ser

WT

pCMV-FLAG-XPC-WT

XP

Xeroderma pigmentosum

XPC

Xeroderma pigmentosum, complementation group C

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TRAF3IP3

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Highlights Four novel mutations of XPC were identified in two unrelated Chinese patients.

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The truncated Q320X XPC abolished GGR function in vivo.

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Readthrough of full length XPC can be induced by G418 treatment in vivo.

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