Bui et al. Human Genome Variation (2020) 7:2 https://doi.org/10.1038/s41439-020-0089-z Human Genome Variation ARTICLE Open Access A novel nonsense mutation of ERCC2 in a Vietnamese family with xeroderma pigmentosum syndrome group D Chi-Bao Bui 1,2, Thao Thi Phuong Duong2,VienTheTran3, Thuy Thanh T. Pham2,TungVu2,GiaCacChau4, Thanh-Niem Van Vo5,VinhNguyen6, Dieu-Thuong Thi Trinh 7,8 and Minh Van Hoang7 Abstract Xeroderma pigmentosum (XP) group D, a severe disease often typified by extreme sun sensitivity, can be caused by ERCC2 mutations. ERCC2 encodes an adenosine triphosphate (ATP)-dependent DNA helicase, namely XP group D protein (XPD). The XPD, one of ten subunits of the transcription factor TFIIH, plays a critical role in the nucleotide- excision repair (NER) pathway. Mutations in XPD that affect the NER pathway can lead to neurological degeneration and skin cancer, which are the most common causes of death in XP patients. Here, we present detailed phenotypic information on a Vietnamese family in which four members were affected by XP with extreme sun sensitivity. Genomic analysis revealed a compound heterozygous mutation of ERCC2 that affected family members and single heterozygous mutations in unaffected family members. We identified a novel, nonsense mutation in one allele of ERCC2 (c.1354C > T, p.Q452X) and a known missense mutation in the other allele (c.2048G > A, p.R683Q). Fibroblasts isolated from the compound heterozygous subject also failed to recover from UV-driven DNA damage, thus recapitulating aspects of XP syndrome in vitro. We describe a novel ERCC2 variant that leads to the breakdown of the 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; NER pathway across generations of a family presenting with severe XP. Introduction XP diagnosis requires both clinical examination and Xeroderma pigmentosum (XP) syndrome, which is genetic analysis. Sethi et al.2 reported that while sun sen- inherited in an autosomal recessive manner, is characterized sitivity in XP correlated with disease severity scoring, half of by sun sensitivity (severe sunburn with irritation and blis- all patients had no history of abnormal sunburn. Genetic tering, dry skin, freckles, hyper- and hypopigmentation) and analysis has revealed a potential mechanism for this type of sunlight-induced ocular symptoms (photophobia, photo- sun sensitivity in selected XP patients through variants in sensitivity, impaired vision, and cataract)1.Approximately nucleotide-excision repair (NER) genes. NER pathway 25% of XP patients have neurological abnormalities, genes, including DDB2, ERCC1, ERCC2, ERCC3, ERCC4, including cognitive decline, loss of reflex and tonus, pro- ERCC5, POLH, XPA,andXPC, eliminate DNA damage by gressive hearing loss, and ataxia. The average life expec- UV light or chemical carcinogens1. In particular, ERCC2 tancy of XP individuals with and without neurological (OMIM: 126340) encodes the XP group D protein (XPD), a degeneration is 29 and 37 years old, respectively1. DNA helicase and subunit of transcription factor TFIIH, which is involved in basal transcription and NER. Thus, mutations in XPD that disrupt NER can lead to XP. Correspondence: Minh Van Hoang ([email protected]) 1Biomedical Research Center, School of Medicine, Vietnam National University, A number of mutations in ERCC2 have previously been Ho Chi Minh City, Vietnam reported to cause XP group D (OMIM: 278730), tri- 2 Functional Genomics Unit, DNA Medical Technology, Ho Chi Minh City, chothiodystrophy 1 (OMIM: 601675), and cere- Vietnam 3,4 Full list of author information is available at the end of the article. brooculofacioskeletal syndrome 2 (OMIM: 610756) . These authors contributed equally: Chi-Bao Bui, Thao Thi Phuong Duong © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Japan Society of Human Genetics Bui et al. Human Genome Variation (2020) 7:2 Page 2 of 8 Notably, a 4-bp deletion at nucleotides 668–671 and a MarkDuplicates by PICARD and Base Quality Score S541 mutation in ERCC2 were reported in a Japanese XP Recalibration by GATK. VCF files were generated with patient5. Another study described a -2 frame shift muta- GATK Haplotype Caller and filtered by GATK Variant tion with a loss of two Ts at nucleotides 1781–1782 on Filtration, SNP (QD < 2.0, FS > 60.0, MQ < 40.0, MQ one allele, a deletion of nucleotides 1823–1825 (AGA), RankSum < −12.5, Read Pos Rank-Sum < −8.0) and and an insertion of TTTCGG at this site on the other INDEL (QD < 2.0, FS > 200.0, Read Pos Rank-Sum < allele6. The same study also described another patient −20.0). Finally, ANNOVAR was used to intersect variant with R112H and L485P mutations6. A further mutation of annotations from UCSC RefSeq, dbSNP 150, gnomAD, R616Q has been reported in one individual, albeit pre- ESP6500, ExAC, 1000G, and dbNSFP v3.5. senting with a milder sun sensitivity7. However, the most For two of the patients, II-4 and II-6, we generated common mutations found in XP patients are the R683Q/ 28,864,756 and 24,816,804 raw reads; 97% and 95.9% – W variants7 11. reads mapped to the reference genome with at least 10× Herein, we describe three siblings with extreme sun coverage after preprocessing; average depths of 59× (96% sensitivity who carry the p.R683Q missense mutation in above 10×) and 66× (95% above 10×) on captured exome one allele but also have a novel p.Q452X nonsense intervals, respectively. mutation in the other allele of the ERCC2 gene. These patients, captured within a genetic analysis across three Variant prioritization and Sanger sequencing generations of the same family, provide further insights We applied an in-house bioinformatics pipeline for the into the molecular basis of XP. WES analysis. Rare variants with MAF < 0.01 in several population databases were selected for further analysis. Materials and methods dbNSFP was utilized to identify the top candidate var- Phenotypes of affected individuals iants by compiling prediction scores from 29 algorithms Three patients (II-4, II-6, and II-10) were first recruited (SIFT, SIFT4G, Polyphen2-HDIV, Polyphen2-HVAR, to the Medical University Center 3 in 2013. These patients LRT, MutationTaster2, MutationAssessor, FATHMM, were all siblings of a family living in Tay Ninh Province, MetaSVM, MetaLR, CADD, VEST4, PROVEAN, Vietnam. Physicians recorded a detailed phenotypic ana- FATHMM-MKL coding, FATHMM-XF coding, fitCons, lysis of each patient to include dermatology, ophthal- LINSIGHT, DANN, GenoCanyon, Eigen, Eigen-PC, mology, and neurology pathologies based on the XP M-CAP, REVEL, MutPred, MVP, MPC, PrimateAI, Disease Severity Scoring System2. Family members pro- GEOGEN2, and ALoFT) and 9 conservation scores vided individual, written informed consent for undergoing (PhyloP × 3, phastCons × 3, GERP++, SiPhy and bSta- a clinical assessment and detailed genetic analysis and tistic). The family segregation of all detected variants was being photographed for publication purposes under the validated by Sanger sequencing. University Medical Center ethics board (15 people, 3 generations). Informed consent was not sought for those Unscheduled DNA synthesis (UDS) assay individuals who were deceased (four people). Primary dermal fibroblast cells were isolated from patients and normal individuals following an established Whole-exome sequencing (WES) protocol12. Cells were then seeded on 96-well microtiter Genomic DNA was extracted from peripheral blood plates and cultured for 16 h in FBS-supplemented DMEM. using the QIAamp DNA Blood Mini Kit (#51104, QIA- Half of the plates were irradiated with UVC (UV+), GEN, Venlo, LI, the Netherlands). DNA was examined for whereas the other half were wrapped in aluminum foil quality using DropSense96, Qubit 2.0, and TapeStation. (UV–). Cells were then incubated in serum-free EdU The prepared libraries were sequenced by 2 × 100 bp (Invitrogen, Waltham, MA, USA) supplemented with paired-end sequencing using Agilent SureSelect Human DMEM immediately after the irradiation per the estab- All Exon V5 (Agilent Technologies, Santa Clara, CA, lished protocol13. Cells were then incorporated with EdU, USA) on a NovaSeq 6000 Sequencing System (Illumina, fixed in 4% paraformaldehyde, and treated with Alexa San Diego, CA, USA) by Macrogen company in Seoul, Fluor 488-azide coupling solution (Invitrogen, Waltham, Republic of Korea. Using paired FASTQ reads, FASTQC MA, USA). Nuclei were stained with DAPI (Thermo was used to obtain useful diagnostic scores, including the Fisher Scientific, Waltham, MA, USA). The fluorescence Phred-score distribution along the reads, GC content intensity of Alexa Fluor 488 and DAPI in 25 different distribution, read-length distribution, and sequence fields was calculated for each cell type.