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Comparative Study
. 2008 Aug;128(8):2055-68.
doi: 10.1038/jid.2008.48. Epub 2008 Mar 27.

Xeroderma pigmentosum-variant patients from America, Europe, and Asia

Hiroki Inui  1 , Kyu-Seon Oh, Carine Nadem, Takahiro Ueda, Sikandar G Khan, Ahmet Metin, Engin Gozukara, Steffen Emmert, Hanoch Slor, David B Busch, Carl C Baker, John J DiGiovanna, Deborah Tamura, Cornelia S Seitz, Alexei Gratchev, Wen Hao Wu, Kee Yang Chung, Hye Jin Chung, Esther Azizi, Roger Woodgate, Thomas D Schneider, Kenneth H Kraemer
Affiliations
Comparative Study

Xeroderma pigmentosum-variant patients from America, Europe, and Asia

Hiroki Inui et al. J Invest Dermatol. 2008 Aug.

Abstract

Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. Clinical appearance of XP-V patients
Family A: (a) XP31BE, 60 year Caucasian man had full-thickness skin removed from his face as a teenager because of multiple skin cancers. This skin was replaced with sun-protected skin from his abdomen and has been free of cancers ever since. He had BCC, SCC, and melanomas on non-grafted skin of his head and upper extremities. Family C: (b) C-1, XP71TMA, 8-year-old Turkish boy, had SCC. (c) C-2, XP70TMA, 17-year-old sister of XP71TMA, had SCC and BCC. (d) C-3, XP98TMA, 26-year-old sister of XP71TMA, had a large SCC on her left cheek. Family D: (e) D-1, XP91TMA, a 10-year-old Turkish man, had no skin cancers. (f) D-2, XP92TMA, brother of XP91TMA, died at age 22 years of metastasis of the SCC on his lip. Family F: (g) F-1, XP161BE, a 46-year-old Caucasian woman with multiple skin BCCs and melanomas. (h) F-2, XP164BE, 52-year-old sister of XP161BE, had multiple BCCs, SCCs, and melanomas. Family G: (i) XP3GO, a 30-year-old German man with multiple BCCs, SCCs, and melanomas. Family I: (j) XP224BE, a 31-year-old Caucasian woman, had multiple BCCs and melanomas.
Figure 2
Figure 2. Post-UV host cell reactivation assay showing normal DNA repair in XP-V cells
UV-treated or control unirradiated luciferase containing plasmid was transfected into cells from normal (AG5186) (light gray bar), XP-C (XP108DC) (dark gray bar), and putative XP-V donors (XP31BE, XP38BE, XP164BE, XP70TMA, XP92TMA, XP3GO, XP224BE, and XP10TA) (open bars). Two days later luciferase activity was measured. The relative luciferase activity of the UV treated to the control plasmid reflects repair activity of the cells. The XP-C cell had low activity and the putative XP-V cells had repair activity in the normal range. The mean ± SEM of triplicate experiments is shown.
Figure 3
Figure 3. Semi-quantitative estimation of polη and polι protein in XP and normal fibroblasts
Normal and putative XP-V cells were assayed by IP-Western blotting of whole-cell extracts. (a) Polη protein and (b) polι protein.
Figure 4
Figure 4. Sequence analysis of family A
(a) Genomic DNA of XP31BE shows change of G to C at exon 6 splice donor site (arrows). There is a single curve in the uncloned DNA, indicating homozygous or hemizygous mutation. (b) RT-PCR using RNA from XP31BE with forward primer C504F (in exon 5) and reverse primer C892R (spanning the exon 8/7 junction) showed a single 388-bp band in normal cells and two bands (arrows) at 284 and 346 bp in XP31BE cells. The diagram indicates deletion of the entire 104 bp of exon 6 in the type-I splice variant and partial deletion (42 bp) of exon 6 in the type-II splice variant. (c) PCR-RFLP assay for family A. The G-to-C mutation creates a new DdeI restriction site. The gel shows genomic DNA from the father, XP31BE, mother, and a normal donor. + Indicates DdeI treatment. The arrows indicate 63 and 54-bp bands of digested DNA. * Indicates the normal-size band.
Figure 5
Figure 5. Analysis of genomic DNA of families C and D
The C454T mutation in exon 4 creates a new DdeI restriction site. DNA from a normal donor, affected siblings XP70TMA, XP71TMA, XP98TMA, and their mother (XPH95TMA) in family C, and affected siblings XP91TMA and XP92TMA in family D were separated on 3% agarose gel. + Indicates DNA digested with DdeI. The digested DNA showed two bands of 92 and 132 bp (arrows). * Indicates the normal-size band.
Figure 6
Figure 6. Sequence analysis of families E and F
(a) Genomic DNA from XP38BE in family E shows an insertion of a G in exon 10. There is a single sequence following the insertion indicating that the DNA is homozygous or hemizygous for this frameshift mutation. (b) PCR-RFLP assay of family E. The insertion of G creates a new BsII site in the genomic DNA. Genomic DNA from a normal donor, patients XP38BE, XP39BE, and their mother, XPH253BE, was run on a gel. + Indicates BsII treatment. The 105 and 248-bp bands in the digested DNA are indicated by arrows. * Indicates the normal-size band. (c) PCR-RFLP analysis for family F. Genomic DNA from XP164BE, XP161BE, the husband, son, daughter, mother, and father of XP161BE was run on a gel. + Indicates BsII treatment. The 105 and 248-bp bands in the digested DNA are indicated by arrows. * Indicates the normal size band.
Figure 7
Figure 7. Mutation spectrum and predicted proteins in XP-V cells
The top line shows the 11 exons of POLH genomic sequence on chromosome 6 as filled rectangles. The location of frameshift, nonsense, and missense mutations in each allele for the cells studied is shown above the line and the splice site mutations are shown below the line. The second line shows the mRNA with the ATG initiation codon in exon 2 and the TAG stop codon in exon 11. The 713-amino-acid pol-η protein is shown in the third line. The N-terminal 400 amino acids are highly conserved in Y-family polymerases, and contain the catalytic domain of the polymerase. There is a nuclear localization signal located at amino acids 682–698 (NLS). The C-terminal region from amino acids 628–662 contains a C2H2 zinc finger that is involved in DNA-binding ubiquitin. A PCNA-binding site is located at the extreme C-terminus of the protein. The bottom portion of the figure shows the predicted size of the protein from each allele or splice variant form from the XP-V patients and the description of the mutation at the cDNA and protein level. PCNA, proliferating-cell nuclear antigen.
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