A CIB1 Splice-Site Founder Mutation in Families

LETTER TO THE EDITOR

A CIB1 Splice-Site Founder Mutation in Families with Typical Epidermodysplasia Verruciformis Journal of Investigative Dermatology (2019) -, -e-; doi:10.1016/j.jid.2018.11.011

TO THE EDITOR Epidermodysplasia verruciformis (EV), an autosomal recessive genodermatosis, is characterized by persistent cutaneous infections of human papilloma viruses (HPVs) of the b-genus (de Jong et al., 2018a; Huang et al., 2018). The early clinical manifestations include thin, tinea versicolor-like lesions and flat warts that develop to protruding tumors. Cutaneous malignancies, particularly squamous cell carcinomas, can develop on these lesions, primarily on sunexposed areas. EV is considered typical, or classic, when the b-HPV infection in the skin is an isolated clinical feature and there is no evidence of compromised T cell-mediated immunity. In atypical, or nonclassic, forms of EV, the cutaneous lesions are associated with widespread viral, bacterial, or fungal infections and development of hematologic malignancies due to compromised T-cell immunity (Youssefian et al., 2019). The genetic basis of b-HPV infection in patients with the typical form of EV was initially shown to consist of biallelic mutations in the TMC6 and TMC8 genes, which encode EVER1 and EVER2, respectively (Ramoz et al., 2002; Youssefian et al., 2018). Quite recently, mutations in a third gene, CIB1, which encodes CIB1, with a ubiquitous pattern of expression and pleiotropic functions, have been identified in other patients with typical EV (de Jong et al., 2018b). CIB1, EVER1, and EVER2 form a complex that serves physiologically as a restriction factor in the skin, limiting infections by b-HPVs, which are widespread and asymptomatic in the general population. Thus, the human CIB1EVER1-EVER2 complex governs innate, keratinocyte-intrinsic immunity to bHPVs (de Jong et al., 2018b).

In this study, we report a consanguineous family with three affected individuals with the typical form of EV with a splice junction mutation in CIB1. The consequences of the mutation were investigated with RNA sequencing (RNA-seq) and differential gene expression heatmap analysis, which showed a complex splicing pattern of CIB1 premRNA leading to loss of function and nonsense-mediated mRNA decay. The proband, a 42-year-old female of Iranian origin with Persian ethnicity, was seen in a dermatology clinic for cutaneous findings consisting of numerous, occasionally confluent flat warts on the trunk and extremities, palmar hyperkeratosis, and multiple ulcerated papules and nodules on the extremities (Figure 1). The skin lesions started developing around puberty and had progressively increased in number and size. The proband is alive, with cutaneous lesions and with no evidence of systemic infections or hematologic malignancies. However, she has multiple basal cell and squamous cell carcinomas, which have metastasized to the bones of the lower extremities, necessitating amputation. The proband was the second of three siblings, all healthy at birth but who developed similar cutaneous lesions (Figure 1b). The proband’s older brother (IV-1) died at the age of 18 years of meningitis, and the proband’s younger sister (IV-3) died of complications of metastatic skin cancer after completion of our study. The clinically healthy parents are first cousins once removed (Figure 1b). Histopathology of the flat warts of the skin of the proband showed hyperkeratosis and acanthosis, with keratinocytes displaying coarse keratohyalin granules, perinuclear halo, and blue-gray pallor, characteristic of EV (Figure 1c). The

Abbreviations: EV, epidermodysplasia verruciformis; HPV, human papillomavirus; ROH, regions of homozygosity; RNA-seq, RNA sequencing; SNP, single nucleotide polymorphism; WES, whole exome sequencing Accepted manuscript published online 29 November 2018; corrected proof published online XXX ª 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

proband also had multiple hyperpigmented plaques with verrucous surface and ulcerations and scaling with raised pearly borders. Excisional biopsy samples from these lesions showed the presence of islands and broad anastomosing bands of malignant cells in the dermis with palisading basaloid cells and squamous differentiation and areas of keratinization, consistent with basosquamous carcinoma (Figure 1d). PCR-based HPV genotyping of the skin lesions showed abundance of HPV5 and HPV8. In comparison, no evidence for the presence of HPV6, 20, 21 or 38, or EpsteinBarr virus infection was noted (Figure 1e). To assess the immune phenotype of the proband with EV, analyses of CD4þ and CD8þ T-cell, B-cell, natural killer cell, monocyte, and thymocyte populations in the peripheral blood of the proband (IV-2) were performed on gated fluorescence activated cytometry with 11 cell surface markers (see Supplementary Materials and Methods online). The percentages of these cell types, when compared with isotype controls, showed normal immune cell parameters (see Supplementary Figure S1 online). Collectively, based on clinical findings, histopathology of skin lesions, HPV genotyping, and immunophenotyping, the affected individuals in this family were diagnosed with a typical, genetic form of EV. Whole exome sequencing (WES) was performed on DNA isolated from the peripheral blood of the proband and her younger sister (IV-2 and IV-3 in Figure 1b). Well over 100,000 sequence variants were identified in the DNA of both patients (Figure 1f). Bioinformatics filtering of the annotated variants, which considered exonic/ splicing variants only, was used to remove synonymous sequence variants and examine homozygous variants shared in the DNA of these two patients, reducing the number of annotated variants under consideration to 1,052 (Figure 1f). Subsequent examination of variants with minor allelic www.jidonline.org

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H Vahidnezhad et al. CIB1 Mutations in EV

Figure 1. Clinical features, family pedigree, cutaneous histopathology, HPV typing, and identification of CIB1 mutations with functional consequences at the mRNA level in patients with EV. (a) Widespread distribution of flat warts on the trunk and extremities, as well as palmar hyperkeratosis, in patient IV-2. Patient IV-3 has multiple warts and hyperkeratotic lesions consistent with squamous cell carcinoma on the back of the right hand. (b) Pedigree of the three affected siblings with EV shows consanguinity. The proband is identified by an arrowhead. (c) Histopathology of lesional skin from the proband showed epidermal hyperkeratosis, with keratinocytes showing coarse keratohyaline granules, perinuclear halos, and blue-gray pallor, consistent with HPV infection. (d) Biopsy sample from a verrucous lesion showed infiltrating islands and anastomosing bands of basaloid cells with monomorphic squamous differentiation, consistent with basosquamous carcinoma. (e) Quantitative real-time PCRebased viral analysis in the skin lesions shows abundance of HPV-8 and HPV-5. No evidence of the presence of other HPV strains indicated or EBV infection was noted. (f) Whole exome sequencing followed by filtering with the steps indicated identified CIB1 as the likely candidate gene. (g) Homozygosity mapping with a single nucleotide polymorphism array consisting of 550,000 markers identified a number of ROHs (blue vertical blocks) in DNA from patients IV-2 and IV-3. The positions of candidate genes associated with EV were aligned along the autosome (dashed vertical lines). The position of CIB1 co-aligned with a block of ROH, which was shared by both affected individuals. (h) Sanger sequencing of genomic DNA confirmed the presence of homozygous mutation in this gene. (i) Sanger sequencing of cDNA, (j) RNA-seq, and (k) differential gene expression profiling by heatmap analysis of CIB1 provided evidence for aberrant splicing. (j) A Sashimi plot showed complex splicing of CIB1 pre-mRNA, including deletion of exon 2. (i) The deletion of exon 2 was confirmed by Sanger sequencing of reverse transcription PCR product. (k)

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frequencies of less than 0.001 reduced the number of candidate genes to 80. Next, to identify potential candidate genes, homozygosity mapping with a single nucleotide polymorphism (SNP) array consisting of 550,000 markers was performed (Vahidnezhad et al., 2018a; Vahidnezhad et al., 2018b) (see Supplementary Materials and Methods). This approach identified several regions of homozygosity (ROHs) of more than 2 megabase-pairs (Mb), and five genes with homozygous mutations were located in ROHs shared with both affected individuals (Figure 1g, and see Supplementary Table S1 online). Only one of these genes, CIB1, is known to be associated with immune functions (de Jong et al., 2018b). Furthermore, alignment of known EV-associated gene loci with the homozygosity map showed that CIB1 was the only gene co-aligning with shared ROHs in both patients, thus identifying it, in the context of the patients’ phenotype, as the candidate gene in this family. Careful examination of the WES BAM files, assisted by Integrated Genomic Viewer (IGV; Broad Institute and Regents of the University of California), showed that CIB1 harbored a splice site mutation, c.52-2A>G, at the intron 1/ exon 2 border. The mutation was confirmed by Sanger sequencing of genomic DNA (Figure 1h). This mutation co-segregated in the family, with both affected individuals examined being homozygous for the mutation, while their parents were heterozygous carriers with no clinical evidence of EV. This mutation, which we also identified in another Iranian family with EV (de Jong et al., 2018b), was predicted by the GenScan algorithm (Burge and Karlin, 1997) to eliminate the acceptor site of exon 2 with activation of a cryptic acceptor site in intron 1. This was predicted to result in retention of 319 base pairs of intron 1 and create an open reading =

Heatmap-based expression profiling showed significantly reduced CIB1 mRNA levels compared with nine randomly selected housekeeping genes, indicating nonsensemediated mRNA decay. Chr, chromosome; EBV, Epstein-Barr virus; EV, epidermodysplasia verruciformis; HPV, human papillomavirus; NTC, non-template negative control; ROH, region of homozygosity; RNA-seq, RNA sequencing.

H Vahidnezhad et al. CIB1 Mutations in EV

frame for a 47-amino acid translation product (1e17 from exon 1 and 18e47 from part of intron 1), followed by a premature termination codon. Because the c.52-2A>G mutation in CIB1 was predicted to alter the splicing involving intron 1 and exon 2, two approaches to examine its consequences were undertaken. First, reverse transcription-PCR was performed using RNA isolated from a skin biopsy sample from the proband as a template, and Sanger sequencing of the product identified the absence of exon 2 sequences in a major transcript of the CIB1 mRNA (Figure 1i). Exon 2 consists of 35 nucleotides, and consequently, this deletion was predicted to result in frameshift of translation and truncation of the protein. Second, RNA-seq was performed from RNA isolated from a skin biopsy samples (for details, see Supplementary Materials). In control samples, canonical splicing removing intron 2 was noted with no evidence of alternative splicing (Figure 1j). Sashimi plot of the patient’s mRNA showed a significantly lower number of reads, potentially reflecting a low level of CIB1 transcripts as a result of nonsense-mediated mRNA decay. This interpretation was confirmed by differential gene expression profile of CIB1 compared with nine housekeeping genes, as detected by heatmap analysis, which showed significantly reduced expression of CIB1 (Figure 1k). Also, approximately one third of the transcripts confirmed the deletion of exon 2, and the remaining transcripts showed more complex, aberrant splicing pattern with retention of part of intron 1 sequences, slightly different from that predicted by the in silico analysis (as described) (Figure 1j). The family with EV examined in this study has a homozygous splice site mutation that was proven here to alter the splicing pattern of mutant CIB1 premRNA. We recently identified the same mutation in another family from the same region of Iran consisting of five affected individuals with similar, although somewhat milder, phenotypes with later onset of skin cancers (de Jong et al., 2018b). To examine the relationship of the c.52-2A>G mutation in

these two families, haplotype analysis using SNP markers surrounding the CIB1 locus in chromosome 15, region q26.1, was performed (see Supplementary Figure S2 online). Haplotype analysis showed conservation of these SNPs surrounding the mutation in CIB1 between the two individuals examined in this study (family 1) and four affected individuals in another family (family 2) that we reported previously (de Jong et al., 2018b), indicating founder effect. Recombination events flanking the CIB1 locus in one individual (Family 1, IV-2) reduced the size of the shared haplotype to 2.2 Mb (see Supplementary Figure S2). To estimate the age of the recurrent CIB1 mutation, we selected one member in each family (IV-2, Figure 1b and IV-3, kindred F, Figure 1 in de Jong et al., 2018b) and compared their genotypes to establish a shared haplotype, based on the exome data. A common homozygous haplotype of 1.8 Mb around the CIB1 mutation was identified. The likelihood-based method ESTIAGE (Genin et al., 2004) estimated the age of the most recent common ancestor at 26 generations (95% confidence interval ¼ 8e102.) Considering a generation of 25 years, this leads to a most recent common ancestor who lived 650 (95% confidence interval ¼ 200e2,700) years ago. This study was approved by the institutional review board of the Pasteur Institute of Iran, and the patients gave written informed consent to participate in research and to have their images and medical histories published. The sequence data emanating from WES and RNA-seq analyses were submitted to the Sequence Read Archive (NCBI.nlm.NIH.gov/SRA, submission number SUV4739718). The CIB1 mutation has been deposited in ClinVar (https://submit.ncbi.nlm.nih.gov/subs/ clinvar_file/SUB4534284/, submission identification SUB4621706).

Hassan Vahidnezhad1,2, Leila Youssefian1,3,4, Amir Hossein Saeidian1,4, Behzad Mansoori5,6, Ali Jazayeri7, Arghavan Azizpour8, Kambriz Kamyab Hesari9, Mehdi Yousefi10,11, Sirous Zeinali12,13, Emmanuelle Jouanguy14,15, JeanLaurent Casanova14,15,16,17,18 and Jouni Uitto1,* 1 Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; 2Biotechnology Research Center, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran; 3 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 4Genetics, Genomics and Cancer Biology PhD Program, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; 5Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; 6Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran; 7Department of Information Science, College of Computing and Informatics, Drexel University, Philadelphia, Pennsylvania, USA; 8 Department of Dermatology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran; 9Department of Pathology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran; 10Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran; 11 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; 12 Biotechnology Research Center, Department of Molecular Medicine, Pasteur Institute or Iran, Tehran, Iran; 13Kawsar Human Genetics Research Center, Tehran, Iran; 14Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France; 15 Paris Descartes University, Imagine Institute, Paris, France; 16St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York, USA; 17Pediatric HematologyeImmunology Unit, Necker Hospital for Sick Children, Paris, France; and 18 Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA * Corresponding author e-mail: Jouni.Uitto@ Jefferson.edu

SUPPLEMENTARY MATERIAL

The authors state no conflict of interest.

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2018.11.011.

ACKNOWLEDGMENTS

REFERENCES

The authors thank the patients and their families for participation in this study. Carol Kelly assisted in manuscript preparation.

Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol 1997;268:78e94.

CONFLICT OF INTEREST

www.jidonline.org

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H Vahidnezhad et al. CIB1 Mutations in EV de Jong SJ, Imahorn E, Itin P, Uitto J, Orth G, Jouanguy E, et al. Epidermodysplasia verruciformis: inborn errors of immunity of human beta-papillomaviruses. Front Microbiol 2018a;9:1222. de Jong SJ, Matos I, Crequer A, Hum D, Gunasekharan V, Lorenzo-Diaz L, et al. The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to b-papillomaviruses. J Exp Med 2018b;215: 2289e310. Genin E, Tullio-Pelet A, Begeot F, Lyonnet S, Abel L. Estimating the age of rare disease mutations: the example of Triple-A syndrome. J Med Genet 2004;41:445e9. Huang S, Wu JH, Lewis DJ, Rady PL, Tyring SK. A novel approach to the classification of

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epidermodysplasia verruciformis. Int J Dermatol 2018;57:1344e50.

Exp Dermatol 2018b; https://doi.org/ 10.1111/exd.13501 (accessed December 18, 2018).

Ramoz N, Rueda LA, Bouadjar B, Montoya LS, Orth G, Favre M. Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis. Nat Genet 2002;32:579e81.

Youssefian L, Vahidnezhad H, Mahmoudi H, Saeidian AH, Daneshpazhooh M, Kamyab Hesari K, et al. Epidermodysplasia verruciformis: genetic heterogeneity and EVER1 and EVER2 mutations revealed by genome-wide analysis [e-pub ahead of print] J Invest Dermatol 2018; https://doi.org/10.1016/j. jid2018.07.010 (accessed December 18, 2018).

Vahidnezhad H, Youssefian L, Jazayeri A, Uitto J. Research techniques made simple: genomewide homozygosity/autozygosity mapping is a powerful tool to identify candidate genes in autosomal recessive genetic diseases. J Invest Dermatol 2018a;138:1893e900. Vahidnezhad H, Youssefian L, Saeidian AH, Zeinali S, Touati A, Abiri M, et al. Genome-wide single nucleotide polymorphism-based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa.

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Youssefian L, Vahidnezhad H, Yousefi M, Saeidian AH, Azizpour A, Touati A, et al. Inherited interleukin 2-inducible T-cell (ITK) kinase deficiency in siblings with epidermodysplasia verruciformis and Hodgkin lymphoma. Clin Infect Dis 2019 (in press).