==== Front Biomed Res Int Biomed Res Int BMRI BioMed Research International 2314-6133 2314-6141 Hindawi 10.1155/2020/4215632 Research Article A Novel Missense Variant of TP63 Heterozygously Present in Split-Hand/Foot Malformation Geng Hao 1 2 3 https://orcid.org/0000-0002-9966-3642Tang Dongdong 1 2 3 Xu Chuan 1 2 3 https://orcid.org/0000-0001-5919-8478He Xiaojin hxj0117@126.com 1 2 3 https://orcid.org/0000-0003-1483-3321Zhang Zhiguo zzg_100@163.com 1 2 3 1Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Hefei, 230022 Anhui, China 2NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No. 81 Meishan Road, Hefei, 230032 Anhui, China 3Key Laboratory of Population Health across Life Cycle (Anhui Medical University), Ministry of Education of the People's Republic of China, No. 81 Meishan Road, Hefei, 230032 Anhui, China Academic Editor: Valeria D'Argenio 2020 26 11 2020 2020 421563231 7 2020 29 10 2020 11 11 2020 Copyright © 2020 Hao Geng et al.2020This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Split-hand/foot malformation (SHFM) is a severe congenital disability mainly characterized by the absence or hypoplasia of the central ray of the hand/foot. To date, several candidate genes associated with SHFM have been identified, including TP63, DLX5, DLX6, FGFR1, and WNT10B. Herein, we report a novel variant of TP63 heterozygously present in affected members of a family with SHFM. Methods This study investigated a Chinese family, in which the proband and his son suffered from SHFM. The peripheral blood sample of the proband was used to perform whole-exome sequencing (WES) to explore the possible genetic causes of this disease. Postsequencing bioinformatic analyses and Sanger sequencing were conducted to verify the identified variants and parental origins on all family members in the pedigree. Results By postsequencing bioinformatic analyses and Sanger sequencing, we identified a novel missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 in this family that results in a substitution of methionine with isoleucine, which is probably associated with the occurrence of SHFM. Conclusion A novel missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 in SHFM was thus identified, which may enlarge the spectrum of known TP63 variants and also provide new approaches for genetic counselling of families with SHFM. National Key R&D Program of China2019YFC1005106Anhui Research Institute of Translational Medicine2017zhyx30Natural Science Foundation of Anhui Province1908085QH355 ==== Body 1. Introduction Split-hand/foot malformation (SHFM) is a severe congenital abnormality mainly characterized by the absence or hypoplasia of the central rays of the hand/foot, which can be isolated or syndromic [1]. The reported incidence of SHFM ranges from 1/6000 to 1/20000, worldwide. The incidence in China could be higher, underlying higher disabilities in infants [2, 3]. Genetic and environmental factors have been proven to contribute significantly to the occurrence of congenital malformations. Several candidate genes have been reported to be associated with SHFM, including TP63 (OMIM 603273), DLX5 (OMIM 600028), DLX6 (OMIM 600030), FGFR1 (OMIM 136350), WNT10B (OMIM 601906), and BHLHA9 (OMIM 615416). The majority of SHFM cases display autosomal dominant inheritance, but other modes of inheritance have also been described [4, 5]. In addition, environmental exposure to medication and chemicals also increases the risk of limb malformations [6, 7]. In the present study, we investigated an isolated Chinese family with no history of exposure to environmental risk factors. In this family, the proband and his son suffered from SHFM. Whole-exome sequencing (WES) was used to detect possible genetic lesions, and a novel missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 was identified to be associated with the occurrence of SHFM in this family. 2. Materials and Methods 2.1. Subjects We investigated 3 generations of a Chinese family from Anhui province, with four family members participating in our study. The proband and his son suffered from SHFM. Peripheral blood samples were collected from all family members for genetic analyses. Clinical symptoms and imaging results of the affected individuals were also recorded. All participants signed informed consent, and this study was approved by the local ethics committee. 2.2. WES, Postsequencing Bioinformatic Analyses, and Sanger Sequencing Genomic DNA was extracted from peripheral blood samples taken from all participants using a DNA blood mini kit (Qiagen, Germany). After quality control, the proband's DNA was used for WES, performed by the Beijing Genomics Institute (Shenzhen, China) with a MGISEQ-2000 genetic sequencer. Procedures were described as follows: (1) library prepared and assessed, (2) sequenced by MGISEQ-2000, (3) reads aligned with hg19 using BWM and GATK software after data filtering, (4) variants identified and annotated (1KGP, ExAC_all, gnomAD, OMIM, ClinVar, HGMD, SIFT, PolyPhen-2, and MutationTaster), and (5) variants validated by Sanger sequencing and cosegregation analysis. Detailed method information has been provided in a previous study [8]. 3. Results This study identified a family with two members (II-2 and III-1) diagnosed with SHFM (Figure 1). The proband (II-2), who already had a child with SHFM, went to the reproductive centre for fertility counselling. The proband experienced bilateral split-foot malformations, and his son suffered from cleft hand and foot deformities. No other abnormities were found in the proband or his son. The clinical and imaging features of the affected individuals are shown in Figure 2. Notably, in this family, the proband's father (I-1) died before seeking genetic counselling; thus, the clinical features were not recorded. However, based on descriptions given by his family members, he did not show any clinical signs of limb malformations. Using WES, we identified a novel heterozygous variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 in the proband and his son (Figure 1). This new variant is not found in the gnomAD, 1000G, and ExAC databases (Table 1). An amino acid sequence alignment suggests that the 316th amino acid in TP63 protein is highly conserved among different species (Figure 3(a)). This novel variant was predicted to be disease-causing/probably damaging by MutationTaster and PolyPhen-2 (Table 1). Subsequently, we constructed a partial model of TP63 protein using Swiss-model; the mutated one exhibits an altered three-dimensional structure of TP63 (Figure 3(b)). Finally, Sanger sequencing found this new variant in affected family members but not in healthy individuals, conforming to the cosegregation principle. 4. Discussion SHFM is a severe congenital heterogeneous limb abnormality that mainly affects the development of the central rays in the hand/foot. It may occur in an isolated or syndromic manner. The clinical phenotypes of SHFM are highly variable, ranging from hypoplasia in a single phalanx or syndactyly to aplasia in one or more central limbs [9]. The development of limbs is a very complex process that begins with the formation of limb buds. The apical ectodermal ridge (AER), located at the distal edge of the developing limb bud, acts as the main signal centre regulating growth along the proximal/distal axis. Disruption of the AER may contribute to SHFM [4]. Recently, it has been reported that genetic factors play a crucial role in the occurrence of SHFM. Several chromosomal loci have been identified that associate with the occurrence of SHFM. Chromosomal rearrangements in 7q21 lead to SHFM1; DLX5 and DLX6 located in this area are involved in the development of limb malformation [10, 11]. SHFM2 is caused by mutations in Xq26 [12]. Duplications involving BTRC and FBXW4 in 10q24 contribute to the occurrence of SHFM3 [13, 14]. SHFM4-associated mutations mapping to 3q28 have been found to be in TP63 [15–17]. Dysregulation of the HOXD gene cluster located in 2q31 plays a key role in SHFM5 [18]. WNT10B mutations in 12q13 are involved in the development of SHFM6 [19, 20]. In addition, there exists a specific SHFM with tibia and fibula deficiency called SHFMD. BHLHA9-associated duplications in 17p13 display significant association with SHFMD [21]. SHFM1, 3, 4, and 5 mainly exhibit an autosomal dominant inheritance pattern, while SHFM2 and 6 display X-linked and autosomal recessive models of inheritance, respectively. Heterozygous expression of mutant TP63 could underlie the occurrence of SHFM4 [4, 5]. Hence, it is essential to provide families with histories of SHFM with molecular genetic testing and counselling. In the present study, we identified a novel heterozygous variant of TP63 in an isolated SHFM family. Based on clinical features and WES results, this type was diagnosed as SHFM4, probably inherited in an autosomal dominant inheritance pattern. However, the proband's father died before molecular testing; although he did not show any clinical signs of limb malformations, we cannot exclude paternal inheritance. TP63 is a protein-coding gene comprising 17 exons, 2 promoters, and some variable splice sites. The TP63 isoforms encoded by this gene can be divided into two categories (TAp63 and ΔNp63) whose expression is driven by different promoters. TAp63 isoforms own an N-terminal transactivation (TA) domain, which is absent in ΔNp63 isoforms. Both the TAp63 and ΔNp63 isoforms can be further divided into TAp63 and ΔNp63α, β, and γ variants after undergoing mRNA alternative splicing. TAp63α is the longest isoform, containing a TA domain, a central DNA-binding domain (DBD), an oligomerization domain (OD), a C-terminal Sterile Alpha Motif (SAM), and a Transactivation Inhibitory (TI) domain [22–25]. As a member of the p53 family of transcription factors, TP63 plays a key role in the formation and differentiation of the AER and is crucial to limb development [4]. The newly discovered amino acid substitution (p.Met316Ile) confirmed in this study occurred at a mutational hotspot in DBD, which is responsible for DNA binding. According to the Alamut Visual software and the ACMG 2015 guidelines, this variant is regarded as a class 3-unknown pathogenicity. However, this site in TP63 is evolutionarily highly conserved among different species. Despite there was small physicochemical difference between Met and Ile according to Grantham scores, bioinformatics software (MutationTaster and PolyPhen-2) predicted that this new variant would be disease-causing/probably damaging. Importantly, Swiss-model software also suggested that this novel variant may change the TP63 partial structure in its DNA-binding domain, which may affect the formation and differentiation of the AER, probably leading to limb malformation. In conclusion, a novel heterozygous missense variant (NM_003722.4:c.948G>A; p.Met316Ile) of TP63 was detected in a Chinese family by whole-exome sequencing. It must be included in genetic diagnoses and counselling discussions of families with SHFM. Acknowledgments We thank all subjects who participated in this study. This study was funded by the Natural Science Foundation of Anhui Province in China (No. 1908085QH355), the Anhui Research Institute of Translational Medicine (2017zhyx30), and the National Key R&D Program of China (No. 2019YFC1005106). Data Availability The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. Ethical Approval This study was approved by the Ethics Review Board of the First Affiliated Hospital of Anhui Medical University. Consent Written informed consent was obtained from all patients. Conflicts of Interest The authors declare that they have no conflicts of interest. Authors' Contributions XH and ZZ designed the study. HG, CX, and DT collected the data. HG and DT analyzed the data. HG and DT wrote the paper. All authors have read and approved the final manuscript. Hao Geng and Dongdong Tang contributed equally to this work. Figure 1 Variant of TP63 identified in a Chinese family with SHFM (NM_3722.4:c.948G>A). The proband (II-2) and his son (III-1) were heterozygous for this variant. The red arrow indicates variant information in Sanger sequencing. Abbreviations: SHFM = split-hand/foot malformation; WT = wild type; M = TP63 variant. Figure 2 (a–d) Clinical phenotypes and X-rays of the proband's son (III-1). (e, f) Clinical phenotypes and X-rays of the proband (II-2). Figure 3 (a) The novel variant (p.Met316Ile) is located in the highly conserved site among species. The red letter represents the mutated amino acid; (b) the partial structure of TP63 protein constructed by Swiss-model with red circles emphasizing the changed conformation. WT = wild type. M = TP63 variant. Table 1 TP63 variant (NM_003722.4:c.948G>A; p.Met316Ile) in a Chinese family with SHFM. Gene TP63 DNA change NM_003722.4:c.948G>A (heterozygous) Amino acid alteration p.Met316Ile Variant type Missense Allele frequency 1KGP 0 ExAC_all 0 gnomAD 0 Function prediction MutationTaster Disease causing (1.000) PolyPhen-2 Probably damaging (0.937) SIFT Tolerated (0.074) Abbreviations: SHFM: split-hand/foot malformation; 1KGP: 1000 Genomes Project; ExAC_all: all the data of Exome Aggregation Consortium; gnomAD: the Genome Aggregation Database. ==== Refs 1 Sowińska-Seidler A. Socha M. Jamsheer A. Split-hand/foot malformation-molecular cause and implications in genetic counseling Journal of Applied Genetics 2014 55 1 105 115 10.1007/s13353-013-0178-5 2-s2.0-84893577162 24163146 2 Elliott A. M. Reed M. H. Chudley A. E. Chodirker B. N. Evans J. A. Clinical and epidemiological findings in patients with central ray deficiency: split hand foot malformation (SHFM) in Manitoba, Canada American Journal of Medical Genetics Part A 2006 140 1428 1439 10.1002/ajmg.a.31245 2-s2.0-33745620226 16673359 3 Dai L. Li Y. H. Deng Y. Prevalence of congenital split hand/split foot malformation in Chinese population Journal of Sichuan University 2010 41 320 323 4 Kantaputra P. N. Carlson B. M. Genetic regulatory pathways of split-hand/foot malformation Clinical Genetics 2018 95 132 139 10.1111/cge.13434 2-s2.0-85053032835 30101460 5 Gurrieri F. Everman D. B. Clinical, genetic, and molecular aspects of split-hand/foot malformation: an update American Journal of Medical Genetics Part A 2013 161 11 2860 2872 10.1002/ajmg.a.36239 2-s2.0-84886235462 6 Al-Jobair A. M. Al-Saleem A. I. Possible association between acetazolamide administration during pregnancy and multiple congenital malformations Drug Design Development & Therapy 2016 10 1471 1476 10.2147/dddt.s99561 2-s2.0-84964414665 7 Kang H. Magee C. Mahan C. Pregnancy outcomes among U.S. Gulf War veterans: a population-based survey of 30,000 veterans Annals of Epidemiology 2001 11 7 504 511 10.1016/S1047-2797(01)00245-9 2-s2.0-0034847857 11557183 8 Lam C.-W. Wong K.-S. Leung H.-W. Law C.-Y. Limb girdle myasthenia with digenic RAPSN and a novel disease gene AK9 mutations European Journal of Human Genetics 2017 25 192 199 10.1038/ejhg.2016.162 2-s2.0-85006275584 27966543 9 Jamsheer A. Genetic background of isolated forms of congenital malformations of the hand Medycyna Wieku Rozwojowego 2008 12 3 729 737 19305023 10 Shamseldin H. E. Faden M. A. Alashram W. Alkuraya F. S. Identification of a novel DLX5 mutation in a family with autosomal recessive split hand and foot malformation Journal of Medical Genetics 2012 49 16 20 22121204 11 Ullah A. Ullah M. F. Khalid Z. M. Ahmad W. Novel heterozygous frameshift mutation indistal-less homeobox 5underlies isolated split hand/foot malformation type 1 Pediatrics International 2016 58 12 1348 1350 10.1111/ped.13023 2-s2.0-85006856444 27085093 12 Faiyaz-Ul-Haque M. Zaidi S. H. E. King L. M. Fine mapping of the X-linked split-hand/split-foot malformation (SHFM2) locus to a 5.1-Mb region on Xq26.3 and analysis of candidate genes Clinical Genetics 2005 67 93 97 10.1111/j.1399-0004.2004.00369.x 2-s2.0-19944432702 15617554 13 Lyle R. Radhakrishna U. Blouin J.-L. Split-hand/split-foot malformation 3 (SHFM3) at 10q24, development of rapid diagnostic methods and gene expression from the region American Journal of Medical Genetics Part A 2006 140A 13 1384 1395 10.1002/ajmg.a.31247 2-s2.0-33745602535 14 Sifakis S. Basel D. Ianakiev P. Kilpatrick M. W. Tsipouras P. Distal limb malformations: underlying mechanisms and clinical associations Clinical Genetics 2001 60 3 165 172 10.1034/j.1399-0004.2001.600301.x 2-s2.0-0034775067 11595015 15 van Bokhoven H. Hamel B. C. J. Bamshad M. p63 gene mutations in EEC syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation American Journal of Human Genetics 2001 69 3 481 492 10.1086/323123 2-s2.0-0034892604 11462173 16 Jin J.-Y. Zeng L. Li K. A novel mutation (c.1010G>T; p.R337L) inTP63as a cause of split-hand/foot malformation with hypodontia The Journal of Gene Medicine 2019 21 10, article e3312 10.1002/jgm.3122 2-s2.0-85071460352 31420900 17 Alves L. U. Pardono E. Otto P. A. Mingroni Netto R. C. A novel c.1037C>G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome Genetics and Molecular Biology 2015 38 1 37 41 10.1590/S1415-475738120140125 2-s2.0-84930027403 25983622 18 Dlugaszewska B. Silahtaroglu A. Menzel C. Breakpoints around the HOXD cluster result in various limb malformations Journal of Medical Genetics 2006 43 2 111 118 10.1136/jmg.2005.033555 2-s2.0-32944464214 15980115 19 Ullah A. Gul A. Umair M. Homozygous sequence variants in the WNT10B gene underlie split hand/foot malformation Genetics & Molecular Biology 2018 41 1 1 8 10.1590/1678-4685-gmb-2016-0162 2-s2.0-85045383360 29384555 20 Ugur S. A. Tolun A. Homozygous WNT10b mutation and complex inheritance in split-hand/foot malformation Human Molecular Genetics 2008 17 17 2644 2653 10.1093/hmg/ddn164 2-s2.0-49649122346 18515319 21 Malik S. Percin F. E. Bornholdt D. Mutations affecting the BHLHA9 DNA-binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik-Percin type American Journal of Human Genetics 2014 95 6 649 659 10.1016/j.ajhg.2014.10.012 2-s2.0-84919596990 25466284 22 Celli J. Duijf P. Hamel B. C. J. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome Cell 1999 99 2 143 153 10.1016/S0092-8674(00)81646-3 2-s2.0-0032744735 10535733 23 McGrath J. A. Duijf P. H. Doetsch V. Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63 Human Molecular Genetics 2001 10 3 221 229 10.1093/hmg/10.3.221 11159940 24 Rinne T. Clements S. E. Lamme E. A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp-Hodgkin/Hay-Wells-like syndromes Human Molecular Genetics 2008 17 13 1968 1977 10.1093/hmg/ddn094 2-s2.0-45749128652 18364388 25 Ghioni P. Bolognese F. Duijf P. H. G. van Bokhoven H. Mantovani R. Guerrini L. Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains Molecular and Cellular Biology 2002 22 24 8659 8668 10.1128/MCB.22.24.8659-8668.2002 2-s2.0-0036892018 12446784