Microduplications of 10Q24 Detected in Two Chinese Patients with Split-Hand/Foot Malformation Type 3

Available online at www.annclinlabsci.org 754 Annals of Clinical & Laboratory Science, vol. 47, no. 6, 2017 Microduplications of 10q24 Detected in Two Chinese Patients with Split-hand/foot Malformation Type 3

Rong Xiang1, Ran Du1, Shuai Guo1, Jie-yuan Jin1, Liang-liang Fan1, Ju-yu Tang2, and Zheng-bing Zhou2

1The State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, and 2 Department of Orthopaedics, Xiangya Hospital of Central South University, Changsha, China

Abstract. Split hand/foot malformation (SHFM) is a congenital heterogeneous disorder with prominent limb deficiency. Seven loci have been identified to associate with SHFM, including SHFM1 to SHFM6 and SHFM/SHFLD. SHFM3 is an autosomal dominant disease, of which the pathogenesis is closely related to the genomic rearrangements at 10q24.We described two Chinese patients with the SHFM3 phenotype by high-resolution SNP array technology. We detected a 534kb microduplication at 10q24 encompassing TLX1, LBX1, BTRC and POLL, and a 600kb duplication with TLX1, LBX1, BTRC, POLL, and FBXW4 located. Sequencing analysis did not find any pathogenic mutations in genes within the region detected by SNP Array Analysis. Our findings may offer more evidence for the further mechanism research of limb-specific congenital disease and will give more precise diagnosis to SHFM3 patients.

Key words: Split hand/foot malformation type 3, SNP array, copy number variation.

Introduction [7]. SHFM4 was mapped to 3q27 and is directly linked to mutations in TP63 [8]. Since the homo- Split hand/foot malformation (SHFM, OMIM zygous mutations in WNT10B have been found, #246560) is a limb malformation with prominent SHFM6 was identified as the autosomal recessive clefts of hands and/or feet due to defect of central inheritance of SHFM [9]. However, the effects of digital rays [1]. SHFM is characterized by median SHFM1 and SHFM5 in the SHFM pathogenesis clefts, ectrodactyly, maldevelopment of the meta- are still unknown. carpals/metatarsals and syndactyly [2]. SHFM dis- plays highly various phenotypes between families SHFM3 mostly shows the non-syndromic SHFM, or/and individuals. It may present as an isolated which is usually accompanied with paraxial abnor- entity or a portion of a congenital syndrome. So far, malities [10]. SHFM3 has been mapped to a large SHFM has affected approximately from 1/25,000 region of 10q24, the causative rearrangement cov- to 1/8,500 individuals [3]. ered a region of 0.5 Mb tandem genetic duplication [11]. Nearly 20% of SHFM were caused by SHFM is a heterogeneous disorder caused by mul- the rearrangements at SHFM3 locus [12]. The tiple loci abnormalities, which has been mapped to minimum duplicated interval included BTRC and seven loci including SHFM1 at 7q21, SHFM2 at POLL [13]. In this study, we reported two patients Xq26, SHFM3 at 10q24, SHFM4 at 3q27, with SHFM3 by SNP Array Analysis. We con- SHFM5 at 2q31, SHFM6 at 12q13 and SHFM/ firmed two microduplications on 10q24, likely the SHFLD at 17p13[4-6]. SHFM1, 3 and 4 are auto- critical regions responsible for the SHFM3 somal dominant diseases. The genomic rearrange- phenotype. ments of the SHFM1 at 7q21 can be either associated with isolated or syndromic limb malformation Materials and Methods

Address correspondence to Zheng-bing Zhou; Department of The Review Board of the Second Xiangya Hospital of Orthopaedics, Xiangya Hospital of Central South University, Changsha, China; phone & fax: +86 073182650230; e mail: xy_ the Central South University has approved this research. [email protected]. All related subjects have consented to this study [14].

Figure 1. Phenotype of two SHFM individuals. (A) Case 1, a one-year-old boy with severe defect on all four limbs. Lack of four digits in hands and the feet had a deep midline cleft. (B) Case 2, a two-year-old girl affected deep clefts on his four limbs and deficiency of two digits on hands.

Clinical report. Two patients in this study were diag- the manufacturer’s instructions. The GenomeStudio nosed as SHFM. One patient (case 1), an one-year-old V2011 software was used to analyze the genotypes (hu- boy with severe defect of all four limbs (Figure 1A), lack man genome build 37(Hg19)) and evaluate the experi- of four digits in hand, aplasia of metacarpals and phalan- mental quality. The call rates of the samples are greater ges. Both feet of the first patient presented midline cleft than 99.0% [15]. and ectrodactyly of three toes. The other patient (case 2), a two-year-old girl, exhibited obvious cleft phenotypes Results containing deep clefts on four limbs, accompanied with two digits deficiency on hands (Figure 1B). We explored the genetic abnormities of our two SHFM patients by SNP array analysis. After com- Cytogenetic analysis. Chromosome analysis was per- paring with the Database of Genomic Variant formed with the peripheral blood samples of the patient by conventional G-Banded techniques with 550 bands (DGV), we detected two CNVs at the region of resolution. Five milliliters of peripheral blood was col- 10q24 in the two patients, respectively. A 534kb lected and subjected to lymphocyte culture according to duplication (chr10:102948934-103496300) was standard cytogenetic protocol. found in case 1, containing TLX1, LBX1, BTRC and POLL (Figure 2A). The other was a 600kb du- DNA extraction. Genomic DNA was extracted from plication (chr10:102,841,029-103,455,809) was peripheral blood of all the subjects by using a DNeasy found in case 2, comprising TLX1, LBX1, BTRC, Blood & Tissue Kit (Qiagen, Valencia, CA) as previously POLL and FBXW4 (Figure 2B). described [14]. Discussion Mutation analysis. Since TP63 is the only causative gene for SHFM, we firstly detected the variants in this gene. We searched candidate genes in the regions de- SHFM is a limb ectrodactyly syndrome that exhib- fined by SNP Array analysis. LBX1, BTRC, DPCD, and ited various phenotypes [16]. Previous study proved FBXW4 were performed by polymerase chain reaction that SHFM3 mainly associated with non-syndrom- (PCR). Sequences of the PCR products were determined ic SHFM, which usually displayed defect of the using the ABI 3100 Genetic Analyzer (ABI, Foster City, third digits, atelia of metacarpals/phalanges and CA) [14]. syndactyly. Moreover, SHFM3 showed more severe phenotypes in feet than hands [6]. These two pa- SNP Array Analysis. Using The Human 660W-Quad tients in our study suffered from serious ectrodac- Chip (Illumina, Inc., San Diego, CA) and the Illumina tyly and cleft on four limbs. The typical phenotypes BeadScan genotyping system (Beadstation Scanner) to obtain the signal intensities of probes (SNP) following conformed to the previous studies. 756 Annals of Clinical & Laboratory Science, vol. 47, no. 6, 2017

Figure 2. CHuman 660w-Quad SNP-array analysis of 10q24 duplication in the patients. (A) A 534kb duplication (chr10:102948934-103496300) in case 1, containing TLX1, LBX1, BTRC and POLL. (B) A 600kb duplication (chr10:102,841,029-103,455,809) in case 2, containing TLX1, LBX1, BTRC, POLL and FBXW4.

The locus of SHFM3 was mapped to chromosome proximal-distal axis of the limb skeleton [22]. LBX1 10 by using array-based genome-wide CNV in is highly expressed in the central nervous system many families with isolated SHFM [17]. However, and skeletal muscles, which is thought to connect the mechanism of the microduplication of the piv- with migratory of muscle precursors and keep this otal region inducing the limb defect remains un- potential [23,24]. TLX1 also called HOX11, which known [18]. The previous findings indicated that a encodes T-cell leukemia homeobox protein 1. 440kb submicroscopic duplication was identified TLX1 is highly expressed in zeugopod region to in 10q24 involving LBX1, β-TRCP, POLL, and maintain normal development of ulna and radius DACTYLIN [11]. Another research revealed a simi- [25]. As all of these genes are completely duplicated lar rearrangement in SHFM families, which was a in our SHFM cases, it is probably that the overex- 325kb duplication including BTRC and POLL. It pression of these genes will induce the phenotypes is the narrowest region identified so far [13]. We of SHFM. identified five entire genes in the duplicated seg- In conclusion, we described two SHFM3 patients ments, all of them were related to the development with severe ectrodactyly and midline cleft. We de- of limbs. Our findings are consistent with the pre- tected two duplications at 10q24 mainly contain- vious reports, where the genomic rearrangements of ing BTRC, LBX1, POLL, TLX1, and FBXW4. We SHFM3 frequently occur in 10q24. BTRC is a suggested that these two duplications may be the popular candidate, which plays a pivotal role in causes of two SHFM3 patients. The present find- many pathways including Wnt/β-catenin, SHH, ings of these two duplications not only provide ad- and NF-#B. All of them are participating in limbs ditional evidence to complete the human data of development [19]. Hence, the abnormal of BTCR SHFM3 but also will contribute to more precise expression may connect with the pathogenesis of diagnosis of SHFM3. SHFM3. FBXW4 is a member of the F-box WD- 40 gene family, which can encode ubiquitin ligases Acknowledgements which play an important role in degradation [20]. We thank the patients for participating in this study. We Previously, this protein was deemed to act on keep- thank the State Key Laboratory of Medical Genetics of ing the apical ectodermal ridge (AER) which was a China for technical assistance. This study was supported by the National Natural Science Foundation of China major signaling center to ensure proper develop- (81370394) and the Open-End Fund for the Valuable ment of a limb [21]. Both of LBX1 and TLX1 are and Precision Instruments of Central South University HOX genes which play key roles in the (CSUZC201737). Prothrombin time and immunoglobulin levels 757

References 13. Lyle, R., U. Radhakrishna, J.L. Blouin, S. Gagos, D.B. Everman, C. Gehrig, C. Delozier-Blanchet, J.V. Solanki, U.C. Patel, S.K. 1. Verma, P.K. and A.A. El-Harouni, Review of literature: genes Nath, F. Gurrieri, G. Neri, C.E.Schwartz, and S.E.Antonarakis, related to postaxial polydactyly. Front Pediatr, 2015. 3: p. 8. Split-hand/split-foot malformation 3 (SHFM3) at 10q24, devel- 2. Elliott, A.M., J.A. Evans, and A.E. Chudley, Split hand foot opment of rapid diagnostic methods and gene expression from malformation (SHFM). Clin Genet, 2005. 68(6): p. 501-5. the region. Am J Med Genet A, 2006. 140(13): p. 1384-95. 3. Klar, A.J., Selective chromatid segregation mechanism pro- 14. Du, R., L.L. Fan, M.J. Lin, Z.J. He, H. Huang, Y.Q. Chen, J.J. posed for the human split hand/foot malformation develop- Li, K. Xia, S.P. Zhao, and R. Xiang, Mutation detection in ment by chromosome 2 translocations:A perspective. Dev Biol, Chinese patients with familial hypercholesterolemia. 2015. 408(1): p. 7-13. Springerplus, 2016. 5(1): p. 2095. 4. Duijf, P.H., H. van Bokhoven, and H.G. Brunner, Pathogenesis 15. Luo, C., Y.F. Yang, B.L. Yin, J.L. Chen, C. Huang, W.Z. of split-hand/split-foot malformation. Hum Mol Genet, 2003. Zhang, J. Wang, H. Zhang, J.F. Yang, and Z.P. Tan, 12 Spec No 1: p. R51-60. Microduplication of 3p25.2 encompassing RAF1 associated 5. Dai, L., Y. Deng, N. Li, L. Xie, M. Mao, and J. Zhu, with congenital heart disease suggestive of Noonan syndrome. Discontinuous microduplications at chromosome 10q24.31 Am J Med Genet A, 2012. 158A(8): p. 1918-23. identified in a Chinese family with split hand and foot malfor- 16. Kano, H., K. Kurosawa, E. Horii, S. Ikegawa, H. Yoshikawa, H. mation. 2013. Kurahashi, and T. Toda, Genomic rearrangement at 10q24 in 6. Gurrieri, F. and D.B. Everman, Clinical, genetic, and molecu- non-syndromic split-hand/split-foot malformation. Hum lar aspects of split-hand/foot malformation: an update. Am J Genet, 2005. 118(3-4): p. 477-83. Med Genet A, 2013. 161A(11): p. 2860-72. 17. Li, C.F., K. Angione, and J.M. Milunsky, Identification of 7. Sivasankaran, A., A.Srikanth, P.S. Kulshreshtha, D. Anuradha, Critical Region Responsible for Split Hand/Foot Malformation J.S. Kadandale, and C.R. Samuel, Split Hand/Foot Type 3 (SHFM3) Phenotype through Systematic Review of Malformation Associated with 7q21.3 Microdeletion: A Case Literature and Mapping of Breakpoints Using Microarray Data. Report. Mol Syndromol, 2016. 6(6): p. 287-96. Microarrays (Basel), 2015. 5(1). 8. Sutton, V.R. and H. van Bokhoven, TP63-Related Disorders, 18. Dai, L., Y. Deng, N. Li, L. Xie, M. Mao, and J. Zhu, in GeneReviews(R), R.A. Pagon, et al., Editors. 1993: Seattle Discontinuous microduplications at chromosome 10q24.31 (WA). identified in a Chinese family with split hand and foot malfor- 9. Blattner, A., A.R. Huber, and B. Rothlisberger, Homozygous mation. BMC Med Genet, 2013. 14: p. 45. nonsense mutation in WNT10B and sporadic split-hand/foot 19. Sowinska-Seidler, A., M. Socha, and A. Jamsheer, Split-hand/ malformation (SHFM) with autosomal recessive inheritance. foot malformation - molecular cause and implications in genetic Am J Med Genet A, 2010. 152A(8): p. 2053-6. counseling. J Appl Genet, 2014. 55(1): p. 105-15. 10. Everman, D.B., C.T. Morgan, R. Lyle, M.E. Laughridge, M.J. 20. Sifakis, S., D. Basel, P. Ianakiev, M. Kilpatrick, and P. Tsipouras, Bamshad, K.B. Clarkson, R. Colby, F. Gurrieri, A.M. Innes, J. Distal limb malformations: underlying mechanisms and clinical Roberson, C. Schrander-Stumpel, H. van Bokhoven, S.E. associations. Clin Genet, 2001. 60(3): p. 165-72. Antonarakis, and C.E. Schwartz, Frequency of genomic rear- 21. Dimitrov, B.I., T. de Ravel, J. Van Driessche, C. de Die- rangements involving the SHFM3 locus at chromosome 10q24 Smulders, A. Toutain, J.R. Vermeesch, J.P. Fryns, K. Devriendt, in syndromic and non-syndromic split-hand/foot malforma- and P. Debeer, Distal limb deficiencies, micrognathia syndrome, tion. Am J Med Genet A, 2006. 140(13): p. 1375-83. and syndromic forms of split hand foot malformation (SHFM) 11. de Mollerat, X.J., F. Gurrieri, C.T. Morgan, E. Sangiorgi, D.B. are caused by chromosome 10q genomic rearrangements. J Med Everman, P. Gaspari, J. Amiel, M.J. Bamshad, R. Lyle, J.L. Genet, 2010. 47(2): p. 103-11. Blouin, J.E. Allanson, B. Le Marec, M. Wilson, N.E. 22. Rux, D.R. and D.M. Wellik, Hox Genes in the Adult Skeleton: Braverman, U. Radhakrishna, C. Delozier-Blanchet, A. Novel Functions Beyond Embryonic Development. Dev Dyn, Abbott, V. Elghouzzi, S. Antonarakis, R.E. Stevenson, A. 2016. Munnich, G. Neri, and C.E. Schwartz,A genomic rearrange- 23. Ikegawa, S., Genomic study of adolescent idiopathic scoliosis in ment resulting in a tandem duplication is associated with split Japan. Scoliosis Spinal Disord, 2016. 11: p. 5. hand-split foot malformation 3 (SHFM3) at 10q24. Hum Mol 24. Fernandez-Jaen, A., J. Suela, D.M. Fernandez-Mayoralas, A.L. Genet, 2003. 12(16): p. 1959-71. Fernandez-Perrone, K.R. Wotton, S. Dietrich, C. Castellanos 12. Klopocki, E., S. Lohan, S.C. Doelken, S. Stricker, C.W. Mdel, J.C. Cigudosa, B. Calleja-Perez, and S. Lopez-Martin, Ockeloen, R. Soares Thiele de Aguiar, K. Lezirovitz, R.C. Microduplication 10q24.31 in a Spanish girl with scoliosis and Mingroni Netto, A. Jamsheer, H. Shah, I. Kurth, R. Habenicht, myopathy: the critical role of LBX. Am J Med Genet A, 2014. M. Warman, K. Devriendt, U. Kordass, M. Hempel, A. Rajab, 164A(8): p. 2074-8. O. Makitie, M. Naveed, U. Radhakrishna, S.E. Antonarakis, 25. Swinehart, I.T., A.J. Schlientz, C.A. Quintanilla, D.P. Mortlock, D. Horn, and S. Mundlos, Duplications of BHLHA9 are as- and D.M. Wellik, Hox11 genes are required for regional pat- sociated with ectrodactyly and tibia hemimelia inherited in terning and integration of muscle, tendon and bone. non-Mendelian fashion. J Med Genet, 2012. 49(2): p. 119-25. Development, 2013. 140(22): p. 4574-82.