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Whole-Exome Sequencing Enables Rapid Determination of Xeroderma Pigmentosum Molecular Etiology

  • Oscar Ortega-Recalde,

    Affiliation Unidad de Genética, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia

  • Jéssica Inés Vergara,

    Affiliations Departamento de Dermatología, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia, Unidad de Dermatología, Clínica Carlos Ardila Lulle, Bucaramanga, Colombia

  • Dora Janeth Fonseca,

    Affiliations Unidad de Genética, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia, Departamento de Biología Molecular, Genética Molecular de Colombia, Bogotá, Colombia

  • Xiomara Ríos,

    Affiliations Departamento de Dermatología, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia, Unidad de Dermatología, Clínica Carlos Ardila Lulle, Bucaramanga, Colombia

  • Hernando Mosquera,

    Affiliations Departamento de Dermatología, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia, Unidad de Dermatología, Clínica Carlos Ardila Lulle, Bucaramanga, Colombia

  • Olga María Bermúdez,

    Affiliation Unidad de Genética, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia

  • Claudia Liliana Medina,

    Affiliations Departamento de Dermatología, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia, Unidad de Dermatología, Clínica Carlos Ardila Lulle, Bucaramanga, Colombia

  • Clara Inés Vargas,

    Affiliation Universidad Industrial de Santander, Bucaramanga, Colombia

  • Argemiro Enrique Pallares,

    Affiliations Departamento de Dermatología, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia, Unidad de Dermatología, Clínica Carlos Ardila Lulle, Bucaramanga, Colombia

  • Carlos Martín Restrepo,

    Affiliations Unidad de Genética, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia, Departamento de Biología Molecular, Genética Molecular de Colombia, Bogotá, Colombia

  • Paul Laissue

    paul.laissue@urosario.edu.co

    Affiliations Unidad de Genética, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogotá, Colombia, Departamento de Biología Molecular, Genética Molecular de Colombia, Bogotá, Colombia

Whole-Exome Sequencing Enables Rapid Determination of Xeroderma Pigmentosum Molecular Etiology

  • Oscar Ortega-Recalde, 
  • Jéssica Inés Vergara, 
  • Dora Janeth Fonseca, 
  • Xiomara Ríos, 
  • Hernando Mosquera, 
  • Olga María Bermúdez, 
  • Claudia Liliana Medina, 
  • Clara Inés Vargas, 
  • Argemiro Enrique Pallares, 
  • Carlos Martín Restrepo
PLOS
x

Abstract

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. In some cases, patients are affected by neurological alterations. XP is caused by mutations in 8 distinct genes (XPA through XPG and XPV). The XP-V (variant) subtype of the disease results from mutations in a gene (XPV, also named POLH) which encodes for Polη, a member of the Y-DNA polymerase family. Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features.

In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T>G, p.Y299X) in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology.

Introduction

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder which affects 1/250.000 to 2.3/million individuals in Western countries [1], [2]. Higher incidence has been reported in specific regions, such as Japan (1∶20.000) and Tunisia (1∶10.000) [3], [4]. XP patients are affected by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. Some patients display neurological alterations such as loss of intellectual functioning, progressive hearing loss, neuromuscular degeneration and tumors of the central nervous system [5], [6]. XP is caused by mutations in 8 distinct genes (named XPA through XPG and XPV), which encode proteins that participate, excepting for XPV, in the nucleotide excision repair (NER) molecular cascade [2]. The NER system is responsible for repairing DNA damage resulting from environmental factors, such as UV radiation and carcinogenic chemical molecules [7]. This multistep mechanism excises and eliminates damaged nucleotides followed by filling in of the consequent gap, using the complementary DNA strand as a template. Consequently, patients carrying NER molecular dysfunctions exhibit a wide spectrum of phenotypic features, including XP [8].

The XP-V (variant) subtype of the disease results from mutations in a gene (XPV, also named POLH) that is not involved with the NER pathway. POLH encodes for Polη, a member of the Y-DNA polymerase family, which is implicated in the synthesis of DNA after injury (translesion synthesis process) [9][11]. Indeed, in XP-V cells POLH mutations reduce the ability to replicate DNA after UV exposure [12], [13].

Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features.

In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T>G, p.Y299X) in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology.

Materials and Methods

Patients

Patients (P1-II:2, P2-II:4) are sibs who attended the Dermatology Unit of the Carlos Ardila Lulle Clinic of Bucaramanga (Colombia) (Figure 1). We could not obtain clinical data and biological samples from patient 3 (P3-II:7) but his family indicated that he is affected by a similar phenotype (see below). This study has been approved by the Ethical Committee at Universidad del Rosario and was conducted according to the Declaration of Helsinki Principles. Patients and their parents (C1-father, C2-mother) provided a written consent form to participate in the study, which includes an authorization to publish these case details.

thumbnail
Figure 1. Pedigree of the XP family.

Black symbols refer to affected individuals. Black dots into first generation individuals symbols (I:1 and I:2) represents the POLH c.897T>G mutation at heterozygous state.

http://dx.doi.org/10.1371/journal.pone.0064692.g001

The patients’ parents were reported to be consanguineous (first cousins). P1 is a 38-year-old female who presented numerous sun-induced skin changes since infancy. At the age of 21 years she presented a unique skin lesion, located on the dorsal region of the nose, which was diagnosed as a squamous cell carcinoma. Since then, 4 additional tumors (basal cell carcinomas) have been diagnosed and surgically resected. No antecedents of melanoma or further neoplasias were recorded. At the age of 37 years physical examination of the skin displayed: Fitzpatrick phototype III, xerosis, multiple telangiectasias and hyper and hypo pigmented lenticular macules (2 mm–5 mm). These lesions were especially located on UV exposed regions, such as face, upper limbs, feet, legs and upper thorax. Additionally, she displayed plaques of keratosis located on the face, arms and legs. Palm and soles, scalp, mucosae and nervous system were unaffected.

P2 (P1′s sister) is a 36-year-old individual affected, since early infancy, by significant photosensitivity related to the occurrence of skin pigmented lesions, which develop on the sunlight exposed areas of the body. Between 31–36 years of age, six basal cell and one squamous cell carcinomas were diagnosed. Physical examination of the skin, performed at 36 years of age, displayed similar findings to those observed in her sister, excepting for the presence of 3 lesions suggestive of basal cell carcinoma.

Exome Capture and High-throughput Sequencing

Genomic DNAs from patients (P1 and P2) and their parents (C1 and C2) were extracted from whole blood samples using standard procedures. Three micrograms of genomic DNA from P1, P2 and C1 was made up to 130 µl with nuclease free water and sonicated to fragment DNA into size ranging between 100 base pairs (bp) to 200bp (Covaris S220). The size distribution was checked by running an aliquot of the sample on Agilent HS Bioanalyzer Chip. The fragmented DNA was size selected using Agencourt AMPure XP SPRI beads (Beckman Coulter). Subsequently, genomic DNA libraries were constructed according to the SureSelect Target Enrichment System protocol using SureSelect Library prep kit (Agilent). DNA was subjected to a series of enzymatic reactions that repair frayed ends, phosphorylate the fragments, add a single nucleotideA overhang and ligate adaptors. After ligation, PCR amplification (5 cycles) was performed to enrich the adaptor-ligated fragments. The prepared libraries were then kept for hybridization as outlined in SureSelect Target Enrichment System for SOLiD 5500 Multiplexed Sequencing protocol. Hybridized library fragments were isolated by magnetic capture using Magnetic Streptavidin coated beads. PCR amplification (8 cycles) was carried out to amplify the captured library and cleaned up using AMPure XP beads (Beckman Coulter). An aliquot of the captured library was run on Agilent High Sensitivity Bioanalyzer Chip (Agilent).

Reference Genome and RefSeq Database

We used the human reference genome GRCh37/hg19 for mapping exome-sequencing (Exome-Seq). The RefSeq sequence database downloaded from NCBI on 12 May 2011 was used as our gene model and for determining aminoacid substitutions.

Exome-Seq Processing-Read Mapping, Variant Calling and Effect Determination

Raw data was obtained from the SOLiD5500×l sequencer and subjected to quality check and filtering using SAET (SOLiD Accuracy Enhancement Tool, LifeScope suite). The high quality filtered data were aligned with human reference genome and variants were called against (LifeScope suite). Then, variations (vcf. file) were transferred to local linux machine. Using snpEFF (sourceforge) the filtered variations were classified, based on their function, into distinct categories such as synonymous, non-synonymous, nonsense, missense, insertions, deletions and splice variations.

Results were further compared with the dbSNP database combining NCBI version 129 to db 137. This helped to identify novel variations and potential deleterious effects using snpSIFT/snpEff. Finally, the annotated variations were further reviewed manually. Exome sequencing assays were performed at Genotypic Technology (Bangalore, Karnataka, India).

POLH Direct Sequencing

In P1, P2, C1 and C2 the exon 8 coding sequence of POLH (ENST00000372236) was amplified using exon-flanking oligonucleotides. Amplicons were purified using shrimp alkaline phosphatase and exonulease I, and sequenced with internal primers. Primer sequences and PCR conditions are available on request. The new sequence data has been deposited in the NCBI-dbSNP database under the accession number rs190423114.

Results

We generated 21 GB data for 3 samples for each individual as paired-end, 75 bases forward and 35 bases reverse, and about 76–85% (38.90–43.51 Mb in length) of the targeted bases were covered at 20X coverage, which sufficiently passed our thresholds for calling SNPs and short insertions or deletions (indels). The bases with quality scores above 20 (99% accuracy of a base call) represent over 79–86% of total sequence data.

Exome-Seq processing showed that patients and C1 are respectively homozygous and heterozygous for the POLH c.897T>G (p.Y299X) mutation. Direct sequencing of POLH exon 8 confirmed these findings. We did not find potential etiological non-synonymous variants in any of the other XP genes.

Discussion

At present, XP patients are classified into eight distinct subtypes depending on the occurrence of mutations in specific genes. However, it has been described that ∼6% of XP cases do not carry mutations in XPA-G or XPV, which suggest potential promoter regulatory variants as well as undiscovered etiological genes that could be identified using direct sequencing or NGS technology [5]. In this context, it would be interesting to sequence, via NGS, XP patients from previous reports who lack mutations in XPA through XPG and XPV genes.

Although the majority of subtypes implicate dysfunctions of proteins which participate in the NER molecular pathway, overlapping clinical features among patients have been observed. Furthermore, the clinical presentation of XPV can be similar to that observed in patients carrying XP-NER gene mutations. For instance, although most XP-V, XP-C and XP-E patients (which represent in Europe and the United States 40–58% of all XP cases) lack severe sunburn reactions, some cases display extreme phenotypes [2], [14], [15]. Mutations in XPC and XPE subtypes, which usually do not lead to neurological disease, can present central nervous system abnormalities due genetic and environmental modifier factors [2], [16], [17]. XPV patients (who are rarely affected by neurological abnormalities) can exhibit skin injuries that vary considerably in severity [18].

In this context, selection of a particular candidate gene for direct sequencing remains difficult. In addition, direct sequencing of all XP coding regions from genomic DNA might be challenging as they encompass more than 100 exons (∼25 kilobases). Whole-exome sequencing has led to the identification of a considerable number of pathogenic mutations in monogenic mendelian disorders, including skin pathologies [19][22]. This approach is especially useful for investigating recessive pathologies, as comparative analysis of coding sequence variations between affected and non-affected individuals enable the efficient identification of homozygous causative mutations [23].

Clinically, P1 and P2 are affected by a mild form of the disease since they have been affected by less than 10 skin tumors, which appeared after the age of 20. In addition, since our patients lack neurological dysfunction, we predicted that the disease pathology most likely resulted from coding mutations in XPC, XPE or XPV. Coding regions of these genes encompass 37 exons, which may be sequenced, in a single patient, via direct sequencing or NGS at similar costs. Indeed, at present primer synthesis and direct sequencing of 20 exons using internal oligonucleotides display similar costs to a single exome sequencing experiment. Furthermore, PCR amplification and Sanger sequencing of a significant number of exons may be labour-intensive.

It is important to note that, at present, downstream bioinformatic analysis of sequencing data is considered as the bottleneck of NGS techniques. Whole-genome and exome sequencing generate a considerable amount of data, which has to be quality controlled, filtered and interpreted in a biological context. This analysis implicates significant computational costs and needs a particular expertise. Although distinct bioinformatics tools have been developed in order to analyze and interpret the increasing amount of sequencing data, sensitivity and efficiency to accurately map noisy reads depends of several factors, such as the choice of single or pair-end reads, the use of long or short reads and the sequencing depth [24]. In this context, the setup of a reliable NGS platform takes a considerable period of time and may represent high costs. Additionally, it has been reported that NGS sensitivity may be low to find somatic mutations [25]. However, very recent reports indicate that this approach may display ∼90% of sensitivity to identify germline mutations [26], [27]. In the present report we have used a similar NGS approach to that described in a previous study, which led us to identify a novel gene related to pachydermoperiostosis/myelofibrosis etiology [28].

Our sequencing experiments revealed that the previously unknown POLH p.Y299X mutation is causative of the phenotype. Up to now, more than 50 POLH mutations have been associated with XPV pathogenesis [4], [10], [18], [29][33]. In a significant proportion of cases these mutations lead to the synthesis of mRNA molecules carrying a premature stop codon, which might be degraded by the nonsense-mediated decay (NMD) system [18]. In our case, since the premature stop codon is situated at least 50 nucleotides upstream of an exon-exon boundary, we propose that mutant mRNAs might be recognized and degraded by the NMD machinery [34]. In addition, the POLH c.897T>G mutation is predicted to generate a truncated protein of 299 residues lacking the C-terminal region of the highly conserved N-terminal domain of the protein, which comprises the enzymatic active site [31], [35], [36]. Therefore, as suggested for other nonsense biallelic mutations which truncate the N-terminal protein region, it is highly likely that the POLH- p.Y299X mutant protein lacks biological activity [31].

Taken together, our results demonstrate that whole-exome sequencing is a powerful strategy, which might be used in a near future, for XP molecular etiology determination. Furthermore, they add information on the clinical and molecular characteristics of XPV patients.

Acknowledgments

We thank Prof. Alan Lehmann for critical reading of the manuscript.

Author Contributions

Conceived and designed the experiments: PL. Performed the experiments: OO DJF OMB. Analyzed the data: PL CMR. Wrote the paper: PL. Performed clinical studies: JIV XR HM CLM CIV AEP CMR.

References

  1. 1. Kleijer WJ, Laugel V, Berneburg M, Nardo T, Fawcett H, et al. (2008) Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA repair 7: 744–750 doi:10.1016/j.dnarep.2008.01.014.
  2. 2. Lehmann AR, McGibbon D, Stefanini M (2011) Xeroderma pigmentosum. Orphanet journal of rare diseases 6: 70 doi:–––10.1186/1750–1172–6–70.
  3. 3. Hirai Y, Kodama Y, Moriwaki S-I, Noda A, Cullings HM, et al. (2006) Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population. Mutation research 601: 171–178 doi:10.1016/j.mrfmmm.2006.06.010.
  4. 4. Ben Rekaya M, Messaoud O, Mebazaa A, Riahi O, Azaiez H, et al. (2011) A novel POLH gene mutation in a xeroderma pigmentosum-V Tunisian patient: phenotype-genotype correlation. Journal of genetics 90: 483–487.
  5. 5. Bradford PT, Goldstein AM, Tamura D, Khan SG, Ueda T, et al. (2011) Cancer and neurologic degeneration in xeroderma pigmentosum: long term follow-up characterises the role of DNA repair. Journal of medical genetics 48: 168–176 doi:10.1136/jmg.2010.083022.
  6. 6. DiGiovanna JJ, Kraemer KH (2012) Shining a light on xeroderma pigmentosum. The Journal of investigative dermatology 132: 785–796 doi:10.1038/jid.2011.426.
  7. 7. Leibeling D, Laspe P, Emmert S (2006) Nucleotide excision repair and cancer. Journal of molecular histology 37: 225–238 doi:––10.1007/s10735–006–9041-x.
  8. 8. Cleaver JE, Lam ET, Revet I (2009) Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nature reviews Genetics 10: 756–768 doi:10.1038/nrg2663.
  9. 9. Masutani C, Kusumoto R, Yamada A, Dohmae N, Yokoi M, et al. (1999) The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399: 700–704 doi:10.1038/21447.
  10. 10. Johnson RE, Kondratick CM, Prakash S, Prakash L (1999) hRAD30 mutations in the variant form of xeroderma pigmentosum. Science (New York, NY) 285: 263–265.
  11. 11. Ohmori H, Friedberg EC, Fuchs RP, Goodman MF, Hanaoka F, et al. (2001) The Y-family of DNA polymerases. Molecular cell 8: 7–8.
  12. 12. Lehmann AR, Kirk-Bell S, Arlett CF, Paterson MC, Lohman PH, et al. (1975) Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation. Proceedings of the National Academy of Sciences of the United States of America 72: 219–223.
  13. 13. Cordonnier AM, Lehmann AR, Fuchs RP (1999) Impaired translesion synthesis in xeroderma pigmentosum variant extracts. Molecular and cellular biology 19: 2206–2211.
  14. 14. Anttinen A, Koulu L, Nikoskelainen E, Portin R, Kurki T, et al. (2008) Neurological symptoms and natural course of xeroderma pigmentosum. Brain: a journal of neurology 131: 1979–1989 doi:10.1093/brain/awn126.
  15. 15. Niedernhofer LJ, Bohr VA, Sander M, Kraemer KH (2011) Xeroderma pigmentosum and other diseases of human premature aging and DNA repair: molecules to patients. Mechanisms of ageing and development 132: 340–347 doi:10.1016/j.mad.2011.06.004.
  16. 16. Khan SG, Oh K-S, Emmert S, Imoto K, Tamura D, et al. (2009) XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms. DNA repair 8: 114–125 doi:10.1016/j.dnarep.2008.09.007.
  17. 17. Schäfer A, Hofmann L, Gratchev A, Laspe P, Schubert S, et al. (2013) Molecular genetic analysis of 16 XP-C patients from Germany: environmental factors predominately contribute to phenotype variations. Experimental dermatology 22: 24–29 doi:10.1111/exd.12052.
  18. 18. Inui H, Oh K-S, Nadem C, Ueda T, Khan SG, et al. (2008) Xeroderma pigmentosum-variant patients from America, Europe, and Asia. The Journal of investigative dermatology 128: 2055–2068 doi:10.1038/jid.2008.48.
  19. 19. Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, et al. (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461: 272–276 doi:10.1038/nature08250.
  20. 20. Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, et al. (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proceedings of the National Academy of Sciences of the United States of America 106: 19096–19101 doi:10.1073/pnas.0910672106.
  21. 21. Diggle CP, Parry DA, Logan CV, Laissue P, Rivera C, et al. (2012) Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis. Human mutation 33: 1175–1181 doi:10.1002/humu.22111.
  22. 22. Nakazawa Y, Sasaki K, Mitsutake N, Matsuse M, Shimada M, et al. (2012) Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair. Nature genetics 44: 586–592 doi:10.1038/ng.2229.
  23. 23. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nature reviews Genetics 12: 745–755 doi:10.1038/nrg3031.
  24. 24. Shyr D, Liu Q (2013) Next generation sequencing in cancer research and clinical application. Biological procedures online 15: 4 doi:–––10.1186/1480–9222–15–4.
  25. 25. Löwer M, Renard BY, de Graaf J, Wagner M, Paret C, et al. (2012) Confidence-based somatic mutation evaluation and prioritization. PLoS computational biology 8: e1002714 doi:10.1371/journal.pcbi.1002714.
  26. 26. Chin EL, da Silva C, Hegde M (2013) Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations. BMC genetics 14: 6 doi:–––10.1186/1471–2156–14–6.
  27. 27. Ware JS, John S, Roberts AM, Buchan R, Gong S, et al. (2013) Next generation diagnostics in inherited arrhythmia syndromes: a comparison of two approaches. Journal of cardiovascular translational research 6: 94–103 doi:–––10.1007/s12265–012–9401–8.
  28. 28. Diggle CP, Parry DA, Logan CV, Laissue P, Rivera C, et al. (2012) Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis. Human mutation 33: 1175–1181 doi:10.1002/humu.22111.
  29. 29. Yuasa M, Masutani C, Eki T, Hanaoka F (2000) Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene. Oncogene 19: 4721–4728 doi:10.1038/sj.onc.1203842.
  30. 30. Kannouche P, Broughton BC, Volker M, Hanaoka F, Mullenders LH, et al. (2001) Domain structure, localization, and function of DNA polymerase eta, defective in xeroderma pigmentosum variant cells. Genes & development 15: 158–172.
  31. 31. Broughton BC, Cordonnier A, Kleijer WJ, Jaspers NGJ, Fawcett H, et al. (2002) Molecular analysis of mutations in DNA polymerase eta in xeroderma pigmentosum-variant patients. Proceedings of the National Academy of Sciences of the United States of America 99: 815–820 doi:10.1073/pnas.022473899.
  32. 32. Tanioka M, Masaki T, Ono R, Nagano T, Otoshi-Honda E, et al. (2007) Molecular analysis of DNA polymerase eta gene in Japanese patients diagnosed as xeroderma pigmentosum variant type. The Journal of investigative dermatology 127: 1745–1751 doi:10.1038/sj.jid.5700759.
  33. 33. Gratchev A, Strein P, Utikal J, Sergij G (2003) Molecular genetics of Xeroderma pigmentosum variant. Experimental dermatology 12: 529–536.
  34. 34. Nagy E, Maquat LE (1998) A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends in biochemical sciences 23: 198–199.
  35. 35. Ling H, Boudsocq F, Woodgate R, Yang W (2001) Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107: 91–102.
  36. 36. Boudsocq F, Ling H, Yang W, Woodgate R (2002) Structure-based interpretation of missense mutations in Y-family DNA polymerases and their implications for polymerase function and lesion bypass. DNA repair 1: 343–358.