Gene 546 (2014) 124–128
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Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma Hai-Ping Ke a,b,1, Hu-Ling Jiang b,1, Ya-Su Lv b,1, Yi-Zhou Huang b, Rong-Rong Liu b, Xiao-Ling Chen b, Zhen-Fang Du b, Yu-Qin Luo c, Chen-Ming Xu c, Qi-Hui Fan d, Xian-Ning Zhang b,⁎ a
Department of Biology, Ningbo College of Health Sciences, Ningbo, Zhejiang Province 315100, China Department of Cell Biology and Medical Genetics, Research Center of Molecular Medicine, National Education Base for Basic Medical Sciences, Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310058, China c Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310006, China d Department of Gynaecology and Obstetrics, Ningbo Women and Children's Hospital, Ningbo, Zhejiang Province 315012, China b
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 23 April 2014 Accepted 22 May 2014 Available online 24 May 2014 Keywords: Epidermolytic palmoplantar keratoderma KRT9 gene Mutation Amniocentesis Prenatal genetic diagnosis
a b s t r a c t Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluidDNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epidermolytic palmoplantar keratoderma (EPPK, OMIM: 144200), an autosomal dominant genodermatosis first described by Vorner in 1901, is the most common form of diffuse keratoderma with a worldwide incidence of 2.2 to 4.4 per 100 000 live newborns (Braun-Falco, 2009; Hamm et al., 1988; Lopez-Valdez et al., 2013; Smith, 2003). EPPK is characterized by clinically diffuse, yellow thickening of the skin on the palms and soles with well-circumscribed erythematous borders during the first weeks or months after birth. Histologically, the Abbreviations: AS-PCR, allele-specific PCR; bp, base pair; CVS, chorionic villus sampling; EPPK, epidermolytic palmoplantar keratoderma; KRT1, keratin 1 gene; KRT9, keratin 9 gene; OMIM, Online Mendelian Inheritance in Man; PGD, preimplantation genetic diagnosis. ⁎ Corresponding author at: Department of Cell Biology and Medical Genetics, Research Center of Molecular Medicine, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou, Zhejiang Province 310058, China. E-mail address: [email protected] (X.-N. Zhang). 1 These authors contributed equally to the work presented.
http://dx.doi.org/10.1016/j.gene.2014.05.048 0378-1119/© 2014 Elsevier B.V. All rights reserved.
affected skin shows hyperkeratosis and vacuolar cytolysis of the granular layer, and electron microscopy shows vacuolization of keratinocytes of the granular layer and clumping of keratin filaments (Fuchs-Telem et al., 2013; Itin and Fistarol, 2005). The degree of hyperkeratosis often varies among families and individuals of the same family. Other symptoms like knuckle pads, digital mutilation, and camptodactyly have also been described in some EPPK patients (Du et al., 2011; Lu et al., 2003; Umegaki et al., 2011). The pathogenic gene of EPPK was initially mapped to chromosome 17q12–q21, the locus of the type I acidic keratin cluster, and responsible mutations were mainly detected in the keratin 9 gene (KRT9), which is expressed exclusively in the differentiating skin of the palms and soles (Fu et al., 2014; Leslie Pedrioli et al., 2012; McLean and Moore, 2011; Reis et al., 1994). A small number of reports also implicated the dispersed KRT1 mutations (Grimberg et al., 2009; Terron-Kwiatkowski et al., 2004, 2006). Members of the keratin intermediate-filament superfamily provide structural integrity to epithelial cells (Chamcheu et al., 2012; Moll et al., 1982; Omary et al., 2004). Structurally, all keratins consist of a central α-helical rod domain of 310 amino-acids flanked by
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non-helical head (V1) and tail (V2) domains. The rod domain is divided into 1A, 1B, 2A and 2B segments (Hamada et al., 2013; Steinert et al., 1993). Mutations generally affect the highly-conserved coil 1A region of the α-helical rod domain of keratin 9, a domain thought to be important for keratin heterodimerization (Fu et al., 2014; Shimomura et al., 2010). Although the successful application of KRT9-specific siRNAs to cultured cells and a knock-out mouse model have been reported, there is no satisfactory treatment for EPPK so far (Fu et al., 2014; Leslie Pedrioli et al., 2012). Therefore, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In this study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated Chinese families based on knowledge of the causative mutation of KRT9.
The peripheral blood genomic DNA of 100 unrelated healthy matched controls was also analyzed. Then, the prenatal molecular diagnosis was accomplished by testing for a KRT9 mutation combined with linkage analysis of polymorphic microsatellite DNA markers (D17S1787 and D17S579) flanking KRT9 (17q12–q21) from amniotic-fluid cells. The PCR primer sequences were:
2. Materials and methods
Amniocentesis was performed under ultrasound guidance at 18 weeks of gestation in the Women's Hospital affiliated to Zhejiang University School of Medicine. About 20 ml of amniotic fluid was collected. No complications occurred after the procedure. Fetal genomic DNA was purified directly from 10 ml amniotic fluid using the Puregene DNA purification kit (Qiagen, Valencia, CA, USA) and the suspect DNA fragment was analyzed. The other 10 ml of amniotic fluid was cultured for conventional G-band karyotyping. This study was conducted in conformity with the Declaration of Helsinki and was approved by the Zhejiang University Review Board. Written informed consent was given by all participants.
2.1. Patients Three unrelated southern Chinese EPPK pedigrees from Jiangxi, Anhui and Zhejiang Province, seeking genetic counseling, were investigated. All the patients showed typical EPPK features, without combined knuckle pads, digital mutilation, or camptodactyly. Family 1 was a fourgeneration EPPK pedigree with 15 affected members, and the proband was a 30-year-old woman who had an induced abortion 5 years ago due to fear of producing an affected child (Liu et al., 2012). Family 2 had 4 affected members, and interestingly, the 28-year-old female proband's left hand showed much milder clinical characteristics than her right hand (Figs. 1A and 2A). Family 3 had 16 affected individuals, and the 30-year-old female proband already had a 3-year-old daughter with EPPK (Figs. 1B and 2B). 2.2. Amniotic fluid-DNA-based prenatal diagnosis First, the pathogenic mutations of the three EPPK pedigrees were identified and confirmed by PCR, Sanger DNA sequencing, and allelespecific PCR (AS-PCR) as previously reported (Liu et al., 2012). The AS-PCR primer sequences to confirm the p.Arg163Gln mutation were (variant nucleotide underlined): Forward: 5′-GCA GGA ACT CAA TTC TCA-3′ Reverse: 5′-TGG ATT CCC TGG CTA TTA-3′.
D17S1787: Forward: 5′-GCT GAT CTG AAG CCA ATG A-3′ Reverse: 5′-TAC ATG AAG GCA TGG TCT G-3′. D17S579: Forward: 5′-AGT CCT GTA GAC AAA ACC TG-3′ Reverse: 5′-CAG TTT CAT ACC AAG TTC CT-3′.
3. Results 3.1. Mutation detection DNA sequencing and AS-PCR results revealed a heterozygous mutation of c. 487CNT (p.Arg163Trp) in the affected members of families 1 and 2, and c.488GNA (p.Arg163Gln) in family 3, within exon 1 of KRT9. Both missense mutations were absent from the healthy controls, dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/), and 1000 genomes (www.1000genomes.org/). 3.2. Prenatal diagnosis Direct DNA sequencing of the PCR product amplified from the fetal DNA samples extracted from amniotic fluid indicated that the fetuses
Fig. 1. Pedigrees of EPPK families 2 (A) and 3 (B). Arrows show the probands.
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Fig. 2. Diffuse hyperkeratosis of palms and soles of the probands, characterized by the presence of a well-demarcated erythematous border. (A) III-2 from family 2. (B) III-15 from family 3.
of families 1 and 2 were free of disease, but the fetus of family 3 had unfortunately inherited the p.Arg163Gln mutation (Fig. 3A and C). In samples from the fetuses of families 1 and 2, AS-PCR failed to reveal the variant DNA fragment (Fig. 3B), and the microsatellite markers D17S1787 and D17S579 flanking KRT9 did not show co-segregation with the phenotype of the disease (Supplementary data, Figs. S1–S4), predicting that the fetuses were homozygously normal. Standard karyotyping revealed 46, XX female fetuses. Healthy female babies were subsequently born at full term without any clinical symptoms of EPPK. Postnatal clinical examination and molecular analysis one year later confirmed the diagnosis (Supplementary data, Figs. S5 and S6). In the sample from the fetus of family 3, AS-PCR (Fig. 3D) and haplotype analysis results both verified that he would be affected with EPPK (Supplementary data, Figs. S7–S9). After careful and painful consideration of the consequences, the proband and her family chose to terminate the pregnancy. 4. Discussion In the absence of radical treatment, prevention of genetic disorders is still one of the available options for genetically disadvantaged people. Currently, considerable progress has been made in the prenatal diagnosis of severe skin disorders, such as epidermolytic hyperkeratosis, harlequin ichthyosis, epidermolysis bullosa, pachyonychia congenita, xeroderma pigmentosum group A, Netherton syndrome, and EPPK (Azarian et al., 2006; Chen et al., 2009; Follmann et al., 2013; Muller et al., 2002; Rothnagel et al., 1998; Smith et al., 1999). EPPK is mainly caused by dominant-negative mutations in KRT9 and less often, KRT1. To date, N 26 KRT9 mutations responsible for EPPK have been described in 1A and 2B segments of the α-helix rod domain (Human Intermediate
Filament Database, www.interfil.org). A mutational hotspot exists at codon 163 of KRT9 that encodes the amino-acid arginine, which is often mutated to tryptophan, glutamine or proline (Leslie Pedrioli et al., 2012; Liu et al., 2012). The frequency of p.Arg163Trp is 31.03% in Chinese EPPK patients, consistent with that in other ethnic groups (29.33%) (Liu et al., 2012). This is a non-conservative change consistent with the methyl CpG deamination mechanism of mutation, which accounts for ~ 90% of point mutations in humans (Ahmad and Rao, 1996). Taking advantage of the fact that most EPPK patients carry KRT9 mutations, a fast, reliable and cost-effective PCR method of testing for EPPK was developed in our laboratory (Chen et al., 2009). This will be helpful for confirming the diagnosis and genetic counseling, and make prenatal diagnosis of the disease by direct mutation analysis feasible. The general approach to detecting fetal hereditary disorders is DNA or RNA analysis of fetal cells following chorionic villus sampling (CVS) or amniocentesis (Essawi et al., 2012; Karaca et al., 2013). These diagnostic tests have an accuracy of ~ 99%, yet carry a relative risk of fetal loss due to their invasiveness. Thus, current research emphasis is focused predominantly on noninvasive prenatal diagnostic techniques that analyze fetal cells or cell-free DNA, mRNA, and microRNA in maternal blood (Bustamante-Aragones et al., 2012; Simpson, 2013). However, for DNA-based prenatal testing, an affected fetus can be diagnosed only at the end of the first trimester and it remains a fundamental issue for couples at risk when an affected fetus is detected. So it is necessary for an alternative approach that avoids the need for three months of pregnancy and allows couples an even earlier decision option. Preimplantation genetic diagnosis (PGD) is a relatively new method that uses in vitro fertilization techniques, and allows embryos to be investigated for genetic disorders before they are transferred to the uterus and before
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Fig. 3. DNA sequencing and allele-specific PCR results. (A) In family 2, III-2 (the proband) showed a heterozygous C-to-T transition at nucleotide position 487 (p.Arg163Trp) in KRT9, while her fetus was normal. (B) AS-PCR electrophoresis results of allele-specific PCR product on 1% agarose gel for family 2. A 589-bp band was present in affected members (III-2 and III-4), while it was absent from the fetus (Fet), a healthy member (III-3), and an unrelated control (Nor). Lad, DNA standard ladder. (C) In family 3, both the proband (III-15) and her fetus had the KRT9 mutant c.488GNA (p.Arg163Gln). (D) In family 3, the 280-bp band in AS-PCR was present in the proband (III-15), her affected first-born daughter (IV-9), and the fetus (Fet), while it was absent from a healthy member (III-16) and an unrelated control (Nor).
pregnancy has begun (Renwick and Ogilvie, 2007). But PGD has clear disadvantages: it is difficult, very costly, not widely available, misdiagnosis may occur from allele dropout, some embryos do not survive the biopsy and many people are opposed to its use due to ethical concerns (Brezina et al., 2012). In addition, a report showed that compared with spontaneous conception, in vitro fertilization by intracytoplasmic sperm injection for male infertility is associated with a small but statistically significantly increased risk of mental retardation (Sandin et al., 2013). Therefore, we suggested that prenatal genetic diagnosis by CVS or amniocentesis is a quite acceptable option in the prediction and prevention of EPPK and helps minimize the risk of disease, especially in low- and middle-income countries. Conflict of interest The authors have no conflict of interest to declare. Acknowledgments This study was supported by the National Natural Science Foundation of China (30972644, 30672250, and J1103603) and the Ningbo Natural Science Foundation (2012A610192). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.048. References Ahmad, I., Rao, D.N., 1996. Chemistry and biology of DNA methyltransferases. Crit. Rev. Biochem. Mol. Biol. 31, 361–380.
Azarian, M., Dreux, S., Vuillard, E., et al., 2006. Prenatal diagnosis of inherited epidermolysis bullosa in a patient with no family history: a case report and literature review. Prenat. Diagn. 26, 57–59. Braun-Falco, M., 2009. Hereditary palmoplantar keratodermas. J. Dtsch. Dermatol. Ges. 7, 971–984. Brezina, P.R., Brezina, D.S., Kearns, W.G., 2012. Preimplantation genetic testing. BMJ 345, e5908. Bustamante-Aragones, A., Rodriguez de Alba, M., Perlado, S., et al., 2012. Non-invasive prenatal diagnosis of single-gene disorders from maternal blood. Gene 504, 144–149. Chamcheu, J.C., Wood, G.S., Siddiqui, I.A., et al., 2012. Progress towards genetic and pharmacological therapies for keratin genodermatoses: current perspective and future promise. Exp. Dermatol. 21, 481–489. Chen, X.-L., Xu, C.-M., Cai, S.-R., et al., 2009. Prenatal diagnosis of epidermolytic palmoplantar keratoderma caused by c.T470C (p.M157T) of the keratin 9 gene in a Chinese kindred. Prenat. Diagn. 29, 911–913. Du, Z.-F., Wei, W., Wang, Y.-F., et al., 2011. A novel mutation within the 2B rod domain of keratin 9 in a Chinese pedigree with epidermolytic palmoplantar keratoderma combined with knuckle pads and camptodactyly. Eur. J. Dermatol. 21, 675–679. Essawi, M.L., Al-Attribi, G.M., Gaber, K.R., et al., 2012. Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs). Gene 509, 120–123. Follmann, J., Macchiella, D., Whybra, C., et al., 2013. Identification of novel mutations in the ABCA12 gene, c.1857delA and c.5653–5655delTAT, causing harlequin ichthyosis. Gene 531, 510–513. Fu, D.J., Thomson, C., Lunny, D.P., et al., 2014. Keratin 9 is required for the structural integrity and terminal differentiation of the palmoplantar epidermis. J. Invest. Dermatol. 134, 754–763. Fuchs-Telem, D., Padalon-Brauch, G., Sarig, O., et al., 2013. Epidermolytic palmoplantar keratoderma caused by activation of a cryptic splice site in KRT9. Clin. Exp. Dermatol. 38, 189–192. Grimberg, G., Hausser, I., Muller, F.B., et al., 2009. Novel and recurrent mutations in the 1B domain of keratin 1 in palmoplantar keratoderma with tonotubules. Br. J. Dermatol. 160, 446–449. Hamada, T., Tsuruta, D., Fukuda, S., et al., 2013. How do keratinizing disorders and blistering disorders overlap? Exp. Dermatol. 22, 83–87. Hamm, H., Happle, R., Butterfass, T., et al., 1988. Epidermolytic palmoplantar keratoderma of Vorner: is it the most frequent type of hereditary palmoplantar keratoderma? Dermatologica 177, 138–145. Itin, P.H., Fistarol, S.K., 2005. Palmoplantar keratodermas. Clin. Dermatol. 23, 15–22. Karaca, E., Karakoc-Aydiner, E., Bayrak, O.F., et al., 2013. Identification of a novel mutation in ZAP70 and prenatal diagnosis in a Turkish family with severe combined immunodeficiency disorder. Gene 512, 189–193.
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Leslie Pedrioli, D.M., Fu, D.J., Gonzalez-Gonzalez, E., et al., 2012. Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder. J. Invest. Dermatol. 132, 1627–1635. Liu, W.-T., Ke, H.-P., Zhao, Y., et al., 2012. The most common mutation of KRT9, c.C487T (p.R163W), in epidermolytic palmoplantar keratoderma in two large Chinese pedigrees. Anat. Rec. (Hoboken) 295, 604–609. Lopez-Valdez, J., Rivera-Vega, M.R., Gonzalez-Huerta, L.M., et al., 2013. Analysis of the KRT9 gene in a Mexican family with epidermolytic palmoplantar keratoderma. Pediatr. Dermatol. 30, 354–358. Lu, Y., Guo, C., Liu, Q., et al., 2003. A novel mutation of keratin 9 in epidermolytic palmoplantar keratoderma combined with knuckle pads. Am. J. Med. Genet. A 120A, 345–349. McLean, W.H., Moore, C.B., 2011. Keratin disorders: from gene to therapy. Hum. Mol. Genet. 20, R189–R197. Moll, R., Franke, W.W., Schiller, D.L., et al., 1982. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31, 11–24. Muller, F.B., Hausser, I., Berg, D., et al., 2002. Genetic analysis of a severe case of Netherton syndrome and application for prenatal testing. Br. J. Dermatol. 146, 495–499. Omary, M.B., Coulombe, P.A., McLean, W.H., 2004. Intermediate filament proteins and their associated diseases. N. Engl. J. Med. 351, 2087–2100. Reis, A., Hennies, H.-C., Langbein, L., et al., 1994. Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK). Nat. Genet. 6, 174–179. Renwick, P., Ogilvie, C.M., 2007. Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues. Expert. Rev. Mol. Diagn. 7, 33–43. Rothnagel, J.A., Lin, M.T., Longley, M.A., et al., 1998. Prenatal diagnosis for keratin mutations to exclude transmission of epidermolytic hyperkeratosis. Prenat. Diagn. 18, 826–830.
Sandin, S., Nygren, K.G., Iliadou, A., et al., 2013. Autism and mental retardation among offspring born after in vitro fertilization. JAMA 310, 75–84. Shimomura, Y., Wajid, M., Weiser, J., et al., 2010. Mutations in the keratin 9 gene in Pakistani families with epidermolytic palmoplantar keratoderma. Clin. Exp. Dermatol. 35, 759–764. Simpson, J.L., 2013. Cell-free fetal DNA and maternal serum analytes for monitoring embryonic and fetal status. Fertil. Steril. 99, 1124–1134. Smith, F., 2003. The molecular genetics of keratin disorders. Am. J. Clin. Dermatol. 4, 347–364. Smith, F.J., McKusick, V.A., Nielsen, K., et al., 1999. Cloning of multiple keratin 16 genes facilitates prenatal diagnosis of pachyonychia congenita type 1. Prenat. Diagn. 19, 941–946. Steinert, P.M., Marekov, L.N., Fraser, R.D., et al., 1993. Keratin intermediate filament structure. Crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly. J. Mol. Biol. 230, 436–452. Terron-Kwiatkowski, A., Terrinoni, A., Didona, B., et al., 2004. Atypical epidermolytic palmoplantar keratoderma presentation associated with a mutation in the keratin 1 gene. Br. J. Dermatol. 150, 1096–1103. Terron-Kwiatkowski, A., van Steensel, M.A., van Geel, M., et al., 2006. Mutation S233L in the 1B domain of keratin 1 causes epidermolytic palmoplantar keratoderma with “tonotubular” keratin. J. Invest. Dermatol. 126, 607–613. Umegaki, N., Nakano, H., Mitsuhashi, Y., et al., 2011. Vorner type palmoplantar keratoderma: novel KRT9 mutation associated with knuckle pad-like lesions and recurrent mutation causing digital mutilation. Br. J. Dermatol. 165, 199–201.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma Hai-Ping Ke a,b,1, Hu-Ling Jiang b,1, Ya-Su Lv b,1, Yi-Zhou Huang b, Rong-Rong Liu b, Xiao-Ling Chen b, Zhen-Fang Du b, Yu-Qin Luo c, Chen-Ming Xu c, Qi-Hui Fan d, Xian-Ning Zhang b,⁎ a
Department of Biology, Ningbo College of Health Sciences, Ningbo, Zhejiang Province 315100, China Department of Cell Biology and Medical Genetics, Research Center of Molecular Medicine, National Education Base for Basic Medical Sciences, Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310058, China c Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310006, China d Department of Gynaecology and Obstetrics, Ningbo Women and Children's Hospital, Ningbo, Zhejiang Province 315012, China b
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 23 April 2014 Accepted 22 May 2014 Available online 24 May 2014 Keywords: Epidermolytic palmoplantar keratoderma KRT9 gene Mutation Amniocentesis Prenatal genetic diagnosis
a b s t r a c t Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluidDNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epidermolytic palmoplantar keratoderma (EPPK, OMIM: 144200), an autosomal dominant genodermatosis first described by Vorner in 1901, is the most common form of diffuse keratoderma with a worldwide incidence of 2.2 to 4.4 per 100 000 live newborns (Braun-Falco, 2009; Hamm et al., 1988; Lopez-Valdez et al., 2013; Smith, 2003). EPPK is characterized by clinically diffuse, yellow thickening of the skin on the palms and soles with well-circumscribed erythematous borders during the first weeks or months after birth. Histologically, the Abbreviations: AS-PCR, allele-specific PCR; bp, base pair; CVS, chorionic villus sampling; EPPK, epidermolytic palmoplantar keratoderma; KRT1, keratin 1 gene; KRT9, keratin 9 gene; OMIM, Online Mendelian Inheritance in Man; PGD, preimplantation genetic diagnosis. ⁎ Corresponding author at: Department of Cell Biology and Medical Genetics, Research Center of Molecular Medicine, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou, Zhejiang Province 310058, China. E-mail address: [email protected] (X.-N. Zhang). 1 These authors contributed equally to the work presented.
http://dx.doi.org/10.1016/j.gene.2014.05.048 0378-1119/© 2014 Elsevier B.V. All rights reserved.
affected skin shows hyperkeratosis and vacuolar cytolysis of the granular layer, and electron microscopy shows vacuolization of keratinocytes of the granular layer and clumping of keratin filaments (Fuchs-Telem et al., 2013; Itin and Fistarol, 2005). The degree of hyperkeratosis often varies among families and individuals of the same family. Other symptoms like knuckle pads, digital mutilation, and camptodactyly have also been described in some EPPK patients (Du et al., 2011; Lu et al., 2003; Umegaki et al., 2011). The pathogenic gene of EPPK was initially mapped to chromosome 17q12–q21, the locus of the type I acidic keratin cluster, and responsible mutations were mainly detected in the keratin 9 gene (KRT9), which is expressed exclusively in the differentiating skin of the palms and soles (Fu et al., 2014; Leslie Pedrioli et al., 2012; McLean and Moore, 2011; Reis et al., 1994). A small number of reports also implicated the dispersed KRT1 mutations (Grimberg et al., 2009; Terron-Kwiatkowski et al., 2004, 2006). Members of the keratin intermediate-filament superfamily provide structural integrity to epithelial cells (Chamcheu et al., 2012; Moll et al., 1982; Omary et al., 2004). Structurally, all keratins consist of a central α-helical rod domain of 310 amino-acids flanked by
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non-helical head (V1) and tail (V2) domains. The rod domain is divided into 1A, 1B, 2A and 2B segments (Hamada et al., 2013; Steinert et al., 1993). Mutations generally affect the highly-conserved coil 1A region of the α-helical rod domain of keratin 9, a domain thought to be important for keratin heterodimerization (Fu et al., 2014; Shimomura et al., 2010). Although the successful application of KRT9-specific siRNAs to cultured cells and a knock-out mouse model have been reported, there is no satisfactory treatment for EPPK so far (Fu et al., 2014; Leslie Pedrioli et al., 2012). Therefore, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In this study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated Chinese families based on knowledge of the causative mutation of KRT9.
The peripheral blood genomic DNA of 100 unrelated healthy matched controls was also analyzed. Then, the prenatal molecular diagnosis was accomplished by testing for a KRT9 mutation combined with linkage analysis of polymorphic microsatellite DNA markers (D17S1787 and D17S579) flanking KRT9 (17q12–q21) from amniotic-fluid cells. The PCR primer sequences were:
2. Materials and methods
Amniocentesis was performed under ultrasound guidance at 18 weeks of gestation in the Women's Hospital affiliated to Zhejiang University School of Medicine. About 20 ml of amniotic fluid was collected. No complications occurred after the procedure. Fetal genomic DNA was purified directly from 10 ml amniotic fluid using the Puregene DNA purification kit (Qiagen, Valencia, CA, USA) and the suspect DNA fragment was analyzed. The other 10 ml of amniotic fluid was cultured for conventional G-band karyotyping. This study was conducted in conformity with the Declaration of Helsinki and was approved by the Zhejiang University Review Board. Written informed consent was given by all participants.
2.1. Patients Three unrelated southern Chinese EPPK pedigrees from Jiangxi, Anhui and Zhejiang Province, seeking genetic counseling, were investigated. All the patients showed typical EPPK features, without combined knuckle pads, digital mutilation, or camptodactyly. Family 1 was a fourgeneration EPPK pedigree with 15 affected members, and the proband was a 30-year-old woman who had an induced abortion 5 years ago due to fear of producing an affected child (Liu et al., 2012). Family 2 had 4 affected members, and interestingly, the 28-year-old female proband's left hand showed much milder clinical characteristics than her right hand (Figs. 1A and 2A). Family 3 had 16 affected individuals, and the 30-year-old female proband already had a 3-year-old daughter with EPPK (Figs. 1B and 2B). 2.2. Amniotic fluid-DNA-based prenatal diagnosis First, the pathogenic mutations of the three EPPK pedigrees were identified and confirmed by PCR, Sanger DNA sequencing, and allelespecific PCR (AS-PCR) as previously reported (Liu et al., 2012). The AS-PCR primer sequences to confirm the p.Arg163Gln mutation were (variant nucleotide underlined): Forward: 5′-GCA GGA ACT CAA TTC TCA-3′ Reverse: 5′-TGG ATT CCC TGG CTA TTA-3′.
D17S1787: Forward: 5′-GCT GAT CTG AAG CCA ATG A-3′ Reverse: 5′-TAC ATG AAG GCA TGG TCT G-3′. D17S579: Forward: 5′-AGT CCT GTA GAC AAA ACC TG-3′ Reverse: 5′-CAG TTT CAT ACC AAG TTC CT-3′.
3. Results 3.1. Mutation detection DNA sequencing and AS-PCR results revealed a heterozygous mutation of c. 487CNT (p.Arg163Trp) in the affected members of families 1 and 2, and c.488GNA (p.Arg163Gln) in family 3, within exon 1 of KRT9. Both missense mutations were absent from the healthy controls, dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/), and 1000 genomes (www.1000genomes.org/). 3.2. Prenatal diagnosis Direct DNA sequencing of the PCR product amplified from the fetal DNA samples extracted from amniotic fluid indicated that the fetuses
Fig. 1. Pedigrees of EPPK families 2 (A) and 3 (B). Arrows show the probands.
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Fig. 2. Diffuse hyperkeratosis of palms and soles of the probands, characterized by the presence of a well-demarcated erythematous border. (A) III-2 from family 2. (B) III-15 from family 3.
of families 1 and 2 were free of disease, but the fetus of family 3 had unfortunately inherited the p.Arg163Gln mutation (Fig. 3A and C). In samples from the fetuses of families 1 and 2, AS-PCR failed to reveal the variant DNA fragment (Fig. 3B), and the microsatellite markers D17S1787 and D17S579 flanking KRT9 did not show co-segregation with the phenotype of the disease (Supplementary data, Figs. S1–S4), predicting that the fetuses were homozygously normal. Standard karyotyping revealed 46, XX female fetuses. Healthy female babies were subsequently born at full term without any clinical symptoms of EPPK. Postnatal clinical examination and molecular analysis one year later confirmed the diagnosis (Supplementary data, Figs. S5 and S6). In the sample from the fetus of family 3, AS-PCR (Fig. 3D) and haplotype analysis results both verified that he would be affected with EPPK (Supplementary data, Figs. S7–S9). After careful and painful consideration of the consequences, the proband and her family chose to terminate the pregnancy. 4. Discussion In the absence of radical treatment, prevention of genetic disorders is still one of the available options for genetically disadvantaged people. Currently, considerable progress has been made in the prenatal diagnosis of severe skin disorders, such as epidermolytic hyperkeratosis, harlequin ichthyosis, epidermolysis bullosa, pachyonychia congenita, xeroderma pigmentosum group A, Netherton syndrome, and EPPK (Azarian et al., 2006; Chen et al., 2009; Follmann et al., 2013; Muller et al., 2002; Rothnagel et al., 1998; Smith et al., 1999). EPPK is mainly caused by dominant-negative mutations in KRT9 and less often, KRT1. To date, N 26 KRT9 mutations responsible for EPPK have been described in 1A and 2B segments of the α-helix rod domain (Human Intermediate
Filament Database, www.interfil.org). A mutational hotspot exists at codon 163 of KRT9 that encodes the amino-acid arginine, which is often mutated to tryptophan, glutamine or proline (Leslie Pedrioli et al., 2012; Liu et al., 2012). The frequency of p.Arg163Trp is 31.03% in Chinese EPPK patients, consistent with that in other ethnic groups (29.33%) (Liu et al., 2012). This is a non-conservative change consistent with the methyl CpG deamination mechanism of mutation, which accounts for ~ 90% of point mutations in humans (Ahmad and Rao, 1996). Taking advantage of the fact that most EPPK patients carry KRT9 mutations, a fast, reliable and cost-effective PCR method of testing for EPPK was developed in our laboratory (Chen et al., 2009). This will be helpful for confirming the diagnosis and genetic counseling, and make prenatal diagnosis of the disease by direct mutation analysis feasible. The general approach to detecting fetal hereditary disorders is DNA or RNA analysis of fetal cells following chorionic villus sampling (CVS) or amniocentesis (Essawi et al., 2012; Karaca et al., 2013). These diagnostic tests have an accuracy of ~ 99%, yet carry a relative risk of fetal loss due to their invasiveness. Thus, current research emphasis is focused predominantly on noninvasive prenatal diagnostic techniques that analyze fetal cells or cell-free DNA, mRNA, and microRNA in maternal blood (Bustamante-Aragones et al., 2012; Simpson, 2013). However, for DNA-based prenatal testing, an affected fetus can be diagnosed only at the end of the first trimester and it remains a fundamental issue for couples at risk when an affected fetus is detected. So it is necessary for an alternative approach that avoids the need for three months of pregnancy and allows couples an even earlier decision option. Preimplantation genetic diagnosis (PGD) is a relatively new method that uses in vitro fertilization techniques, and allows embryos to be investigated for genetic disorders before they are transferred to the uterus and before
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Fig. 3. DNA sequencing and allele-specific PCR results. (A) In family 2, III-2 (the proband) showed a heterozygous C-to-T transition at nucleotide position 487 (p.Arg163Trp) in KRT9, while her fetus was normal. (B) AS-PCR electrophoresis results of allele-specific PCR product on 1% agarose gel for family 2. A 589-bp band was present in affected members (III-2 and III-4), while it was absent from the fetus (Fet), a healthy member (III-3), and an unrelated control (Nor). Lad, DNA standard ladder. (C) In family 3, both the proband (III-15) and her fetus had the KRT9 mutant c.488GNA (p.Arg163Gln). (D) In family 3, the 280-bp band in AS-PCR was present in the proband (III-15), her affected first-born daughter (IV-9), and the fetus (Fet), while it was absent from a healthy member (III-16) and an unrelated control (Nor).
pregnancy has begun (Renwick and Ogilvie, 2007). But PGD has clear disadvantages: it is difficult, very costly, not widely available, misdiagnosis may occur from allele dropout, some embryos do not survive the biopsy and many people are opposed to its use due to ethical concerns (Brezina et al., 2012). In addition, a report showed that compared with spontaneous conception, in vitro fertilization by intracytoplasmic sperm injection for male infertility is associated with a small but statistically significantly increased risk of mental retardation (Sandin et al., 2013). Therefore, we suggested that prenatal genetic diagnosis by CVS or amniocentesis is a quite acceptable option in the prediction and prevention of EPPK and helps minimize the risk of disease, especially in low- and middle-income countries. Conflict of interest The authors have no conflict of interest to declare. Acknowledgments This study was supported by the National Natural Science Foundation of China (30972644, 30672250, and J1103603) and the Ningbo Natural Science Foundation (2012A610192). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.048. References Ahmad, I., Rao, D.N., 1996. Chemistry and biology of DNA methyltransferases. Crit. Rev. Biochem. Mol. Biol. 31, 361–380.
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