Identification of novel mutations and reassignment of archival xeroderma pigmentosum group C cell strains from Japanese patients

概要

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Identification of novel mutations and
reassignment of archival xeroderma pigmentosum
group C cell strains from Japanese patients
Kobayashi, Hirotaka
Pozo, Franklin Mayca
Sakai, Wataru
Sato, Kenji
Sugasawa, Kaoru
(Citation)
The Journal of Dermatology,50(3):407-408

(Issue Date)
2023-03

(Resource Type)
journal article

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Accepted Manuscript

(Rights)
This is the peer reviewed version of the following article: [Kobayashi, H, Pozo, FM,
Sakai, W, Sato, K, Sugasawa, K. Identification of novel mutations and reassignment of
archival xeroderma pigmentosum group C cell strains from Japanese patients. J
Dermatol. 2023; 50: 407–408.], which has been published in final form at…
[https://doi.org/10.1111/1346-8138.16623].
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Identification of novel mutations and re-assignment of archival xeroderma pigmentosum
group C cell strains from Japanese patients

Hirotaka Kobayashi1, Franklin Mayca Pozo1, Wataru Sakai1,2, Kenji Sato3, and Kaoru Sugasawa1,2

Biosignal Research Center, Kobe University, Kobe, Japan

1

Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan

2

Department of Dermatology, Hannan Chuo Hospital, Osaka, Japan

3

Correspondence:
Kaoru Sugasawa, Ph.D., Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku,
Kobe, Hyogo 657-8501, Japan.
E-mail: ksugasawa@garnet.kobe-u.ac.jp

1

Xeroderma pigmentosum (XP) is an autosomal recessive disorder, which is characterized by cutaneous
hypersensitivity to sunlight exposure and a predisposition to skin cancer. Eight genetic complementation
groups have been identified for XP: seven groups (XP-A through G) are associated with defects in
nucleotide excision repair (NER), while the remaining one, an XP variant (XP-V) form, is caused by
impairment of translesion DNA synthesis, but not NER 1,2. The complementation group C is one of the
most common forms of XP in the world, so that a large number of pathogenic mutations have been
identified for the responsible gene XPC (https://www.ncbi.nlm.nih.gov/clinvar/?term=XPC[gene]).
Although the overall frequency of XP in Japan is much higher than that in the whole world, XP-C is a
relatively rare form. Only a few XP-C cases have been reported, for most of which the XPC gene
mutation was not identified. Here we describe genetic analyses of skin fibroblasts from three Japanese
XP patients (XP3KA, XP4KA, and XP40OS), which were assigned to XP-C (Clinical information of
the patients is provided in Supporting information).
Mutations of the XPC gene were analyzed by sequencing of genomic PCR and RT-PCR products
and the results are summarized in TABLE 1 (detailed results of sequencing are provided in Supporting
information, FIGURE S1). We identified compound heterozygous XPC mutations in XP3KA cells: one
allele harbored a well-documented, 2-bp (TG) deletion in exon 9 3, and the other lacked the entire exons
12 and 13, both leading to a frameshift. Breakpoints of the novel large (~2.3 kbp) deletion were
identified within introns 11 and 13. On the other hand, XP4KA cells were homozygous for a novel 1-bp
insertion in exon 2, also leading to a frameshift. As expected, the XPC mRNA levels were significantly
reduced in both cell strains most likely due to the nonsense-mediated mRNA decay, while the expression
of XPC protein was not detected by immunoblot analysis (see Supporting information, FIGURE S2).
Therefore, these results indicate that both patients showed typical null-like phenotypes, which are
common to most of the XP-C cases so far reported. Intriguingly, both XP3KA and XP4KA cells were
homozygous for Ala499Val polymorphism (see Supporting information, FIGURE S3), which could be
implicated in the development of melanoma in both patients 4.
Surprisingly, we failed to identify any pathogenic XPC mutation in XP40OS cells. Therefore,
complementation with the known XP-related genes was re-examined, which revealed that the ERCC2
(XPD) gene, but not XPC, could restore UV-induced unscheduled DNA synthesis in XP40OS cells (see

2

Supporting information, FIGURE S4). Sequencing of the ERCC2 genomic locus revealed the presence
of compound heterozygous mutations (TABLE 1). One allele harbored a base substitution at the second
nucleotide in exon 22, resulting in an amino acid substitution (Arg683Gln) that has been frequently
found in Japanese XP-D cases 5. The other had a novel deletion encompassing last 23 nucleotides of
exon 21. From RT-PCR analysis, we identified at least two types of transcripts expressed from this
second allele, both of which causes a frameshift and potentially encodes mutant XPD proteins of larger
size than the wild-type protein. However, immunoblot analyses detected only the XPD protein of normal
size (see Supporting information, FIGURE S2), indicating that the unrelated C-terminal amino acid
sequences destabilize the mutant XPD proteins in cells. Therefore, it is likely that only the XPD
Arg683Gln protein is relevant to the phenotypes of XP40OS. Based on these results, we conclude that
XP40OS should be re-assigned as XP-D. Indeed, this patient exhibited exaggerated sunburn 2 months
after birth 6, which is characteristically observed symptoms in XP-D, but not in XP-C.
In this study, we identified three novel pathogenic mutations of XP-related genes (two in XPC, and
one in ERCC2), which could be specific for Japanese XP cases. Recently next generation sequencing
has become a powerful approach for diagnosis of various hereditary diseases, especially for detection
of known mutations in responsible genes. It is important for future diagnosis to accumulate data of
pathogenic mutations from analysis of individual cases.

ACKNOWLEDGEMENT

We thank Prof. Chikako Nishigori (Kobe University) for the collection of and discussion on clinical
information as well as critical reading of the manuscript. We also thank Dr. Yuka Nakazawa and Prof.
Tomoo Ogi (Nagoya University) for the complementation tests of XP40OS cells, and Dr. Takashi
Mochizuki (Kanazawa Medical University) for clinical information and discussion on the XP3KA and
XP4KA patients.

3

CONFLICTS OF INTEREST

None declared.

REFERENCES

1. DiGiovanna JJ, Kraemer KH. Shining a light on xeroderma pigmentosum. J Invest Dermatol. 2012;
132: 785–796.
2. Nishigori C, Sugasawa K. DNA Repair Disorders. Singapore: Springer Nature; 2019.
3. Rekaya MB, Messaoud O, Talmoudi F, Nouira S, Ouragini H, Amouri A, et al. High frequency of
the V548A fs X572 XPC mutation in Tunisia: implication for molecular diagnosis. J Hum Genet. 2009;
54: 426–429.
4. Asadian F, Niktabar SM, Ghelmani Y, Kargar S, Akbarian E, Emarati SA, et al. Association of XPC
polymorphisms with cutaneous malignant melanoma risk: evidence from a meta-analysis. Acta Medica.
2020; 63: 101–112.
5. Ono R, Masaki T, Pozo FM, Nakazawa Y, Swagemakers SMA, Nakano E, et al. A 10-year followup of a child with mild case of xeroderma pigmentosum complementation group D diagnosed by wholegenome sequencing. Photodermatol Photoimmunol Photomed. 2016; 32: 174–180.
6. Sato K, Ikenaga M, Sano S. Kinetic analysis of polyethylene glycol-induced cell fusion in cultured
human fibroblasts: Its application to genetic complementation analysis of xeroderma pigmentosum. Med
J Osaka Univ. 1982; 33: 19–28.

4

TABLE 1 Summary of mutations identified in this study.

cell line

mutated
gene

XP3KA

XPC

mutation identified

predicted protein change

allele 1

c.1643_1644delTG

2-bp deletion in exon 9 (ref. 3)

XPC, p.Val548Alafs*25

allele 2

c.2116_2420del
(g.33142_35462del)

deletion of exons 12 and 13

XPC, p.Met706Aspfs*2

c.218_219insT

1-bp insertion in exon 2

XPC, p.Lys73Asnfs*9

XP4KA

XPC

allele 1/2

XP40OS

ERCC2

allele 1

c.2048G>A

single base substitution in exon 22 (ref. ...

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