Genetic Analysis of Preaxial Polydactyly: Identification of Novel Mutations and the Role of ZRS Duplications in a Chinese Cohort of 102 cases

Genetic Analysis of Preaxial Polydactyly: Identification of Novel Mutations and the Role of ZRS Duplications in a Chinese Cohort of 102 cases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Analysis of Preaxial Polydactyly: Identification of Novel Mutations and the Role of ZRS Duplications in a Chinese Cohort of 102 cases Siyu Pu, Zhibo Wang, Xueyang Tang, Daoxi Wang, Xiaodong Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4718373/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2024 Read the published version in Human Genetics → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Preaxial polydactyly (PPD) is a congenital limb malformation, previously reported to be caused primarily by mutations in the ZRS and upstream preZRS regions. This study investigated genetic variations associated with PPD, focusing on point mutations and copy number variations (CNVs) in the ZRS and preZRS regions. Methods: Comprehensive genetic analyses were conducted on 102 patients with PPD, including detailed clinical examinations and Sanger sequencing of the ZRS and preZRS regions. Additionally, real-time quantitative PCR (qPCR) was used to detect CNVs in the ZRS region. The evolutionary conservation and population frequencies of identified mutations were also evaluated. Results: Six point mutations were identified, including four novel mutations with potential pathogenicity, namely, 93G > T (chr7:156584477), 106G > A (chr7:156584464), 278G > A (chr7:156584292), and 409A > C (chr7:156585378). Additionally, qPCR analysis revealed that 66.67% of patients exhibited ZRS duplications. Notably, these duplications were also present in cases with newly identified potential pathogenic point mutations. These findings suggest the possible interaction of point mutations in ZRS and preZRS through a common pathogenic mechanism, leading jointly to PPD. Conclusion: The findings expand the mutation spectrum associated with non-syndromic polydactyly and highlight that, despite different classifications, anterior polydactyly caused by mutations in ZRS and nearby regions may share common pathogenic mechanisms. The incorporation of various mutation types in genetic screening can effectively enhance the rate of pathogenic mutation detection and contribute to the cost-effectiveness of genetic testing for limb developmental defects, thereby promoting healthy births. Preaxial Polydactyly SHH ZRS preZRS Figures Figure 1 Introduction Polydactyly, characterized by bifurcation of digit ray(s) along the longitudinal axis progressing from distal to proximal ends, represents one of the most common congenital limb developmental defects (LDDs)(Evanson et al. 2016 ). It can occur in isolated cases (non-syndromic polydactyly) or be associated with other clinical features (syndromic polydactyly), resulting in a highly heterogeneous phenotype. Based on the Temtamy-McKusick classification, non-syndromic polydactyly can be categorized into preaxial (on the radial side), postaxial (on the ulnar side), and complex types(Malik 2014 ). Due to the significant functional impact of thumb deformities, preaxial polydactyly (PPD) warrants special attention(Blum et al. 2021 ). PPD includes polydactyly of the thumb/hallux (type I, PPD1), the triphalangeal thumb (TPT, type II, PPD2), the index finger (type III), and polysyndactyly or crossed polydactyly (CP, type IV)(Malik 2014 ). PPD1 (OMIM-174400) is the most common PPD subtype and is characterized by the presence of additional digits, more commonly in the hand than the foot(Malik et al. 2014 ). PPD2 (OMIM-174500) presents with an extra phalangeal bone in the thumb and is relatively rare, often occurring concurrently with other phenotypes(Potuijt et al. 2019 ). The etiology of PPD1 and PPD2 is associated with the ectopic expression of Sonic Hedgehog ( SHH ) in the Zone of Polarizing Activity (ZPA), regulated by the ZPA Regulatory Sequence ( ZRS ), located on chromosome 7 (coordinates: chr7:156,583,781 − 156,584,569, reference genome Hg19)(Williamson et al. 2016a ). Mutations in the ZRS gene leading to ectopic expression of SHH in limb buds can result in PPD(Lettice et al. 2014 ; Lohan et al. 2014 ). Recent studies have also found that point mutations in the preZRS sequence (chr7:156,585,155–156,585,786), located upstream of ZRS , are associated with PPD(Potuijt et al. 2018 ; Potuijt et al. 2022 ). Previous studies have screened for pathogenic genes in populations with digit malformations, leading to the identification of several novel mutation sites(Furniss et al. 2009 ; Rao et al. 2018 ; Xiang et al. 2017 ). However, these screenings, which have focused primarily on point mutations, have resulted in very low detection rates. Therefore, the present study focused on PPD1 and PPD2. Analysis of point mutations and copy number variations (CNVs) in 102 affected individuals not only identified four novel mutation sites but also highlighted the significant contribution of ZRS fragment duplications in the pathogenesis of PPD. Thus, the combination of point mutations and CNVs can significantly improve the detection rate of genetic variants in future screenings for PPD1 and PPD2. Patients and Method Patients This study recruited 102 participants from West China Hospital of Sichuan University and Shanghai Ninth People's Hospital. Each case was accompanied by comprehensive X-ray documentation and confirmed for digit malformations by a consensus of two orthopedic surgeons, with classification according to the Temtamy–McKusick schema. Furthermore, cognitive assessments, along with evaluations of craniofacial morphology, stature, foot, and finger lengths, were systematically conducted for all subjects to meticulously exclude concurrent congenital anomalies, thereby ensuring a homogenous cohort comprising solely non-syndromic pre-axial polydactylies. Detailed personal and familial medical histories were meticulously recorded, and blood specimens were procured from both probands and their pedigrees based on their individual genetic profiles. This study was approved by the Institutional Review Board of West China Hospital of Sichuan University with the reference number 2022 Review NO.1623. Written informed consent was obtained from all individual participants recruited in the study or their legal guardians Mutations reported By reviewing existing literature, we conducted a comprehensive summary of reported point mutations within the ZRS and preZRS regions associated with PPD1 and PPD2 (Table 1 ). Additionally, we summarized information on copy number variations encompassing the ZRS fragment that have been linked to PPD 1 and PPD2 (Table 2 ). Table 2 . Previously reported ZRS duplications causing limb development defects. Reference Lohan et al. [2014] Lohan et al. [2014] Lohan et al. [2014] Wieczorek et al. [2009] Wu et al. [2009] Dai et al. [2013] Sun et al. [2008] Lohan et al. [2014] Xu et al. [2019] Xu et al. [2020] Mutation ref 16kb 47kb 75kb 89 kb 97 kb 115 kb 160kb 179 kb 180 kb 182kb Hg19 Chr7: 156,578,108-156,594,751 Chr7: 156,563,856-156,610,632 Chr7: 156,570,780-156,646,750 Chr7: 156,572,751-156,661,877 Chr7: 156,547,469-156,644,074 Chr7: 156,505,616-156,620,919 Chr7: 156,539,605-156,699,998 Chr7: 156,491,887-156,671,016 Chr7: 156,554,005-156,736,751 Chr7: 156,554,005-156,736,751 Preaxial polydactyly + + + + + + + + + + Triphalangeal thumb - - - + + + + + + Partial or complete syndactyly + + + + + + - + + + Feet affected + + + + + + - + - - Reference Lohan et al. [2014] Sun et al. [2008] Wieczorek et al. [2009] Zlotina et al. [2020] Sun et al. [2008] Sun et al. [2008]; Sun et al. [2008] Sun et al. [2008] Klopocki et al. [2008] Liu et al. [2008] Mutation ref 255 kb 265 kb 293 kb 300 kb 334 kb 378kb 437kb 459kb 589kb 0.29Mb Hg19 Chr7: 156,437,229-156,692,706 Chr7: 156,354,085-156,619,399 Chr7: 156,368,541-156,661,877 Chr7: 156,385,810-156,684,811 Chr7: 156,354,085-156,687,613 Chr7: 156,241,020-156,619,399 Chr7: 156,241,020-156,677,759 Chr7: 156,241,020-156,699,998 Chr7: 156,143,386-156,732,204 Chr7: 156,484,201-156,772,643 Preaxial polydactyly + + + + + + + + + + Triphalangeal thumb + + + + + + + + + + Partial or complete syndactyly + + + - - + + + + - Feet affected - - + + - - - - + - Sanger Sequencing Genomic DNA was isolated from whole blood using an TIANamp Blood DNA Kit (DP348-03, TIANGEN, Beijing, China) following the manufacturer’s instructions. Subsequently, primers were designed to amplify the sequences of the ZRS and preZRS genes using polymerase chain reaction (PCR), followed by Sanger sequencing. The primer sequences are listed in Table 3 . PCR was performed in a total volume of 25 µL per reaction, containing 12.5 µL 2×Hieff PCR Master Mix (10102ES03, YEASEN, Wuhan, China), 1 µL genomic DNA (~ 50 ng), and 0.5 µl of each primer (5 µM). Reactions were carried out in a T100 Thermal Cycler (Bio-Rad, Hercules, the USA) with the following conditions: 95℃ for 3 min followed by 35 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 60 s. Table 4 . Clinical feature in polydactylies of this study Polydactyly n Upper Limb (N) Lower Limb (N) Family history Right Left Both Right Left Both Preaxial-I 96 53 34 9 0 0 2 2 Preaxial-II 6 1 2 3 0 0 1 0 TOTAL 102 54 36 12 0 0 3 2 Table 5 . The bioinformatic prediction of the variants/mutations identified gene Change Location Variant ID Frequency conservation ClinVar ZRS 3C>G chr7: 156584567 rs10254391 common YES benign 93G>T chr7:156,584,477 - - NO - 106G>A chr7:156,584,464 rs1354594327 rare NO - 278G>A chr:156,584,292 rs1395888355 rare YES - preZRS 409 A>C chr: 156,585,378 - - YES - 603G>C chr: 156,585,184 rs6949624 common NO benign Table 6 . Summary of the RCN of ZRS region in this cohort using 1.3 as the cut-off value RCN* ≥1.3 <1.3 n (%) 68 (66.7) 34 (33.3) *RCN, Relative copy number Real-time quantitative PCR (qPCR) Based on the ΔΔCT method, qPCR was employed to confirm the duplication of fragments containing the ZRS sequence. Primers were designed using the NCBI Primer-BLAST tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) with reference to previous reports by Sun et al.(Sun et al. 2008 ) (Table 3 ). Each qPCR reaction had a total volume of 10 µL, consisting of 5 µL 2X Hieff qPCR SYBR Green Master Mix (11201ES08, YEASEN, Wuhan, China), 1 µL genomic DNA (~ 10 ng), and 0.2 µL of each primer (2 µM), with triplicates for each sample. Reactions were performed on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, USA), with the following cycling conditions: 95℃ for 3 min and 40 cycles of 95℃ for 15 s, followed by 60℃ for 30 s. After the final cycle, melt curve analysis was conducted to confirm amplification specificity. The experiments were replicated three times. A cut-off Relative Copy Number (RCN) of 1.3 was used to define duplication(Sun et al. 2008 ). Bioinformatic Analysis DNA variants were characterized and compared with those reported in public single nucleotide polymorphism databases ( https://www.ncbi.nlm.nih.gov/snp ), UCSC ( http://genome.ucsc.edu/ ) and the 1000 Genomes dataset ( http://browser.1000genomes.org/index.html ) were used for identification of SNP and conservation of noncoding sequences. Results Clinical features A comprehensive clinical examination was conducted on the 102 patients included in this study; the clinical features are summarized in Table 4 . All the patients exhibited normal intelligence, facial features, body morphology, and hearing. Among the patients, 96 (94.12%) were diagnosed with PPD1 and 6 (5.88%) with PPD2. Bilateral hand involvement was observed in 12 cases (11.76%), while 90 cases (88.24%) presented with polydactyly in only one hand (right: 54 cases, 52.94%; left: 36 cases, 35.29%). Additionally, three patients (2.94%) exhibited bilateral foot involvement. These instances of polydactyly were predominantly sporadic, with only two cases showing familial inheritance. The study also included other affected individuals from these families. Point mutations Sanger sequencing of preZRS and ZRS in the 102 patients identified six point mutations: 3C > G (chr7:156584567), 93G > T (chr7:156584477), 106G > A (chr7:156584464), 278G > A (chr7:156584292), 409A > C (chr7:156585378), and 603G > C (chr7:156585184) (Fig. 1 ). Of these identified variants, 3C > G (rs10254391) and 603G > C (rs6949624) were common genetic polymorphisms observed in the multiple cases, with 46 (45.10%) heterozygous for 3C > G and 38 (37.25%) homozygous for this variant. Similarly, for the 603G > C variant, 43 cases (42.16%) were heterozygous and 47 (46.08%) were homozygous. Both variants are classified as benign according to ClinVar. Mutations, while 106G > A (rs1354594327) and 278G > A (rs1395888355) are classified as rare SNPs, and 93G > T and 409A > C have no prior records. Conservation analysis revealed that the nucleotides at positions 156584477G, 156584464G, and 156585184G were not evolutionarily conserved, whereas 156584567C, 156584292G, and 156585378A were conserved (Table 5 ). Duplication mutations Only four patients were found to have potentially pathogenic point mutations, representing a relatively low frequency. To investigate whether ZRS duplications caused PPD in these 102 patients, primers targeting the region 738-858bp (chr7: 156585427 − 156585307) upstream of ZRS were designed and the RCNs were determined. The qPCR results showed that 68 of the 102 patients had RCN values greater than 1.3 (range: 1.31–2.88), defining them as duplication-positive (66.67%; Tables 6 ). Additionally, all the patients with non-polymorphic SNP point mutations detected by Sanger sequencing were also duplication-positive (93G > T: 1.31; 106G > A: 1.79; 278G > A: 1.92; 409A > C: 1.74). Discussion Limb development disorders (LDDs) are the second most common congenital developmental defects after congenital heart disease(Dolk et al. 2010 ). LDDs have thus been a major focus of research and serve as classical models for the study of embryonic morphogenesis and signal regulation(Zhang et al. 2023 ). Although LDDs are reported to be caused by a combination of genetic and environmental factors(Alexander et al. 2016 ), many of the pathogenic mutations responsible for these malformations remain unidentified(Malik 2014 ), and even when specific genes have been found to be causative, only a small number of cases exhibit mutations in these genes(Lopez-Rios 2016 ). For instance, this study focused on PPD, for which previous research has identified various point mutations within the ZRS and preZRS sequences as causative for PPD1 and PPD2(Potuijt et al. 2018 ; Potuijt et al. 2022 ; Xu et al. 2019 ). However, intensive screening of affected populations has revealed that only a few cases carry these point mutations. This extremely low detection rate significantly impacts patient diagnosis and treatment(Furniss et al. 2009 ; Rao et al. 2018 ; Xiang et al. 2017 ). The ZRS gene is a well-studied limb-specific enhancer that regulates SHH , a morphogen associated with the ZPA, and which plays a crucial role in the anterior-posterior axis development of the limb buds(Williamson et al. 2016b ). Consequently, mutations in ZRS lead to anterior-posterior limb abnormalities, manifesting as incorrect polarity from the thumb to the fifth digit, affecting both the digit number and pattern(Lettice et al. 2014 ; Tickle and Towers 2017 ). Point mutations in ZRS have been confirmed to be associated with PPD1(Malik 2014 ). Additionally, microduplications involving the ZRS region have been implicated in conditions such as TPT, Haas type syndactyly, and Laurin-Sandrow syndrome(Lohan et al. 2014 ; Wieczorek et al. 2010 ; Xu et al. 2020 ). In addition, research by Sagai et al. (Sagai et al. 2005 ) demonstrated that homozygous deletions of ZRS resulted in truncation of the distal limbs in mice. Recent studies have also highlighted the significance of the preZRS region, a 632 bp region located 585 bp upstream of ZRS (Park et al. 2008 ; Xiang et al. 2017 ). Point mutations in preZRS have been identified as causative for both PPD1 and PPD2, indicating a shared pathogenic mechanism between these phenotypes(Potuijt et al. 2022 ). Mutations in preZRS have similarly been shown to disrupt the regulation of SHH expression, leading to defective anterior-posterior limb patterning(Potuijt et al. 2018 ). Both PPD1 and PPD2 are caused by mutations in ZRS and preZRS and exhibit similar phenotypes, namely, preaxial abnormalities. Limb malformations are characterized by reduced penetrance and variable expressivity, resulting in different phenotypes within the same family due to differential expression(Duan et al. 2022 ; Shen et al. 2018 ). Thus, it is hypothesized that PPD1 and PPD2 may represent phenotypes of the same underlying pathogenic mechanism with different expressivity. The present study included primarily sporadic cases of unilateral PPD1, with few accompanying foot deformities. These findings align with the known distribution patterns of PPD1 and PPD2. The results indicated a slightly higher incidence of right-hand deformities compared to left-hand, although the SHH -ZPA-based pathogenic mechanism does not predispose to one side, suggesting that this might be a coincidental observation. In this study, four novel potentially pathogenic point mutations were identified. Among these, both the 278G > A mutation within ZRS and the 409A > C mutation within preZRS show strong evolutionary conservation. Furthermore, the 278G > A mutation is a low-frequency SNP, indicating a strong likelihood of pathogenicity. However, a significant number of cases showed ZRS duplications and elevated RCN values were observed in all four cases with the novel point mutations. It is thus hypothesized that point mutations in both ZRS and preZRS and ZRS duplications contribute to a common pathogenic mechanism leading to PPD1 and PPD2. This suggests that ZRS duplications are pathogenic not only for PPD2 but also for PPD1. Therefore, when investigating the causes of PPD1 and PPD2, it is recommended to screen for point mutations in both ZRS and preZRS , as well as for ZRS duplications. However, no pathogenic cause was identified in 34 cases in this study, indicating that there are still other unknown etiologies awaiting discovery. For example, in 2021, Jin et al. (Jin et al. 2021 ) identified two novel variants in GLIS1 in PPD1 patients, linking GLIS1 to PPD1. This study has some limitations, potentially due to potential selection bias as patient enrolment was confined to PPD1 and PPD2 cases. Additionally, the detection of ZRS duplications was performed using qPCR, which is a relative, rather than an absolute, quantification method, potentially leading to less precise results. Overall, the findings not only expand the spectrum of mutations associated with non-syndromic polydactyly but also suggest that similar phenotypes caused by the same gene may share common pathogenic mechanisms. The incorporation of various mutations in genetic screening tests can effectively enhance the detection rate of pathogenic mutations and also contribute to the cost-effectiveness of genetic testing for LDDs. Declarations Acknowledgment We would like to thank Dr. Xiaoli Wang from Shanxi Provincial Children's Hospital for providing samples used in this study. We also extend our gratitude to the pediatric surgery nursing team at West China Hospital of Sichuan University for their generous assistance in sample collection. Additionally, we appreciate the support provided by the Stem Cell Biology Laboratory and other members of the Laboratory of Pediatric Surgery, West China Hospital, Sichuan University for their generous help in methodology and reagents. Author contributions : Jing Chen and Bin Wang conceived the study and designed the experiments. Siyu Pu conducted the molecular experiments and wrote the manuscript. Jing Chen, Siyu Pu, and Zhibo Wang contributed to data interpretation. Zhibo Wang, Bin Wang, Xueyang Tang, Xiaodong Yang, Jun Jiang, Daoxi Wang, and Yifan Deng performed the clinical assessments and collected the samples. Bo Xiang, Jiayin Yang, and Jing Chen supervised the study. Jing Chen provided critical revisions. All authors read and approved the final manuscript. Funding: This research was supported by National Key R&D Program of China (2022YFC2703700, 2022YFC2703704), National Natural Science Foundation of China (32170813), Science and Technology Department of Sichuan (2024NSFSC0651), and 1·3·5 project for disciplines of excellence–Clinical Research Fund, West China Hospital, Sichuan University (2024HXFH035 & ZYGD23026). Declaration of interests: The authors have no conflicts of interests or financial disclosures that are relevant to the research or information in this paper. Data Availability: The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request. Consent to publish: The authors affirm that human research participants provided informed consent for publication of the images in Figure 1b. References Alexander PG, Clark KL, Tuan RS (2016) Prenatal exposure to environmental factors and congenital limb defects. 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Development 143: 2994-3001. doi: 10.1242/dev.139188 Xiang Y, Jiang L, Wang B, Xu Y, Cai H, Fu Q (2017) Mutational screening of GLI3, SHH, preZRS, and ZRS in 102 Chinese children with nonsyndromic polydactyly. Dev Dyn 246: 392-402. doi: 10.1002/dvdy.24488 Xu C, Yang X, Zhou H, Li Y, Xing C, Zhou T, Zhong D, Lian C, Yan M, Chen T, Liao Z, Gao B, Su D, Wang T, Sharma S, Mohan C, Ahituv N, Malik S, Li Q-Z, Su P (2019) A novel ZRS variant causes preaxial polydactyly type I by increased sonic hedgehog expression in the developing limb bud. Genetics in Medicine 22. doi: 10.1038/s41436-019-0626-7 Xu J, Wu J, Teng X, Cai L, Yuan H, Chen X, Hu M, Wang X, Jiang N, Chen H (2020) Large duplication in LMBR1 gene in a large Chinese pedigree with triphalangeal thumb polysyndactyly syndrome. American Journal of Medical Genetics Part A 182. doi: 10.1002/ajmg.a.61757 Zhang B, He P, Lawrence JEG, Wang S, Tuck E, Williams BA, Roberts K, Kleshchevnikov V, Mamanova L, Bolt L, Polanski K, Li T, Elmentaite R, Fasouli ES, Prete M, He X, Yayon N, Fu Y, Yang H, Liang C, Zhang H, Blain R, Chedotal A, FitzPatrick DR, Firth H, Dean A, Bayraktar OA, Marioni JC, Barker RA, Storer MA, Wold BJ, Zhang H, Teichmann SA (2023) A human embryonic limb cell atlas resolved in space and time. Nature. doi: 10.1038/s41586-023-06806-x Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 24 Oct, 2024 Read the published version in Human Genetics → Version 1 posted Editorial decision: Revision requested 17 Sep, 2024 Reviews received at journal 31 Jul, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 14 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers invited by journal 12 Jul, 2024 Editor assigned by journal 11 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 10 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4718373","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335355409,"identity":"36de319c-2eba-4421-b743-6f4af8fa3ca7","order_by":0,"name":"Siyu Pu","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Pu","suffix":""},{"id":335355410,"identity":"a4067d62-34b7-4cc6-8971-ee02c22cd988","order_by":1,"name":"Zhibo Wang","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai JiaoTong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhibo","middleName":"","lastName":"Wang","suffix":""},{"id":335355411,"identity":"9bfe19c3-b99e-4a61-bf00-7fc5935eb392","order_by":2,"name":"Xueyang Tang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xueyang","middleName":"","lastName":"Tang","suffix":""},{"id":335355412,"identity":"e823d548-354a-4637-84ac-36b95eb8e151","order_by":3,"name":"Daoxi Wang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Daoxi","middleName":"","lastName":"Wang","suffix":""},{"id":335355413,"identity":"0e0427c6-fbc4-423e-8e60-46d118f2a556","order_by":4,"name":"Xiaodong Yang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Yang","suffix":""},{"id":335355414,"identity":"851df05d-3817-4199-9b81-a9cde04b1c38","order_by":5,"name":"Jun Jiang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Jiang","suffix":""},{"id":335355416,"identity":"3f249379-aa14-42f9-9168-ca771e0f754e","order_by":6,"name":"Yifan Deng","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Deng","suffix":""},{"id":335355417,"identity":"bd047016-4b78-4928-bced-31f9149abd6b","order_by":7,"name":"Bo Xiang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Xiang","suffix":""},{"id":335355419,"identity":"e1ac1b55-9639-4631-a4a2-80c5962739a3","order_by":8,"name":"Jiayin Yang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jiayin","middleName":"","lastName":"Yang","suffix":""},{"id":335355421,"identity":"3627b32c-7993-49e6-86c3-a37eee585d4a","order_by":9,"name":"Bin Wang","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai JiaoTong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""},{"id":335355422,"identity":"a579d1a6-9016-4956-be1c-22b37a384bd4","order_by":10,"name":"Jing Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACxmYQ0cDAwA/hM5OgRbKBWC0QfUDlBgeI1cLcznv4xc8dNnnG5w8/k2CosE5sYD97gIDD+NIse8+kFZvdSDOTYDiTntjAk5dAQAuPmTFj2+HEbTd42CRAjAYJHgNitPxP3Nx/BqjlH3FajB8zth1I3MCQA9TSQKQtjL1tyYkzbqQZWyQcSzdu48nBr8Ww/4zxh59tdon9/Ycf3vhQYy3bz36GgJYGBjYJOC8BiNnwqgcCeWDUfCCkaBSMglEwCkY4AADn2UJ4G9aQUgAAAABJRU5ErkJggg==","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-07-10 13:36:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4718373/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4718373/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00439-024-02709-7","type":"published","date":"2024-10-24T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62218532,"identity":"b6a7658a-8b5c-4c55-b81f-e2417050e756","added_by":"auto","created_at":"2024-08-11 12:03:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":483673,"visible":true,"origin":"","legend":"\u003cp\u003eScreening for point mutations in 102 patients using Sanger sequencing.\u003cstrong\u003e a: \u003c/strong\u003eThe 3C\u0026gt;G mutation was found in 84 patient, with 38 cases being homozygous and 46 cases heterozygous. The 603G\u0026gt;C mutation was found in 90 patients, with 47 cases being homozygous and 43 cases heterozygous. \u003cstrong\u003eb:\u003c/strong\u003e The four new point mutations showed heterozygosity, and the X-rays of these patients indicated PPD1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4718373/v1/ad6d034fb5a2445dafdb699a.png"},{"id":67682777,"identity":"5a8581cd-25c7-4264-85e8-1f5bd274b3e1","added_by":"auto","created_at":"2024-10-28 16:15:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1143821,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4718373/v1/646c6886-dd61-4a88-9ff0-424d336ed2d5.pdf"},{"id":62218530,"identity":"4ad0e02d-e84b-4489-8086-560a35f696a3","added_by":"auto","created_at":"2024-08-11 12:03:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26071,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4718373/v1/b15c778bb7546f13dbdbdcf9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenetic Analysis of Preaxial Polydactyly: Identification of Novel Mutations and the Role of ZRS Duplications in a Chinese Cohort of 102 cases\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolydactyly, characterized by bifurcation of digit ray(s) along the longitudinal axis progressing from distal to proximal ends, represents one of the most common congenital limb developmental defects (LDDs)(Evanson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It can occur in isolated cases (non-syndromic polydactyly) or be associated with other clinical features (syndromic polydactyly), resulting in a highly heterogeneous phenotype. Based on the Temtamy-McKusick classification, non-syndromic polydactyly can be categorized into preaxial (on the radial side), postaxial (on the ulnar side), and complex types(Malik \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the significant functional impact of thumb deformities, preaxial polydactyly (PPD) warrants special attention(Blum et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PPD includes polydactyly of the thumb/hallux (type I, PPD1), the triphalangeal thumb (TPT, type II, PPD2), the index finger (type III), and polysyndactyly or crossed polydactyly (CP, type IV)(Malik \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). PPD1 (OMIM-174400) is the most common PPD subtype and is characterized by the presence of additional digits, more commonly in the hand than the foot(Malik et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). PPD2 (OMIM-174500) presents with an extra phalangeal bone in the thumb and is relatively rare, often occurring concurrently with other phenotypes(Potuijt et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe etiology of PPD1 and PPD2 is associated with the ectopic expression of Sonic Hedgehog (\u003cem\u003eSHH\u003c/em\u003e) in the Zone of Polarizing Activity (ZPA), regulated by the ZPA Regulatory Sequence (\u003cem\u003eZRS\u003c/em\u003e), located on chromosome 7 (coordinates: chr7:156,583,781\u0026thinsp;\u0026minus;\u0026thinsp;156,584,569, reference genome Hg19)(Williamson et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e). Mutations in the \u003cem\u003eZRS\u003c/em\u003e gene leading to ectopic expression of \u003cem\u003eSHH\u003c/em\u003e in limb buds can result in PPD(Lettice et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lohan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Recent studies have also found that point mutations in the \u003cem\u003epreZRS\u003c/em\u003e sequence (chr7:156,585,155\u0026ndash;156,585,786), located upstream of \u003cem\u003eZRS\u003c/em\u003e, are associated with PPD(Potuijt et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Potuijt et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have screened for pathogenic genes in populations with digit malformations, leading to the identification of several novel mutation sites(Furniss et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, these screenings, which have focused primarily on point mutations, have resulted in very low detection rates. Therefore, the present study focused on PPD1 and PPD2. Analysis of point mutations and copy number variations (CNVs) in 102 affected individuals not only identified four novel mutation sites but also highlighted the significant contribution of \u003cem\u003eZRS\u003c/em\u003e fragment duplications in the pathogenesis of PPD. Thus, the combination of point mutations and CNVs can significantly improve the detection rate of genetic variants in future screenings for PPD1 and PPD2.\u003c/p\u003e"},{"header":"Patients and Method","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eThis study recruited 102 participants from West China Hospital of Sichuan University and Shanghai Ninth People\u0026apos;s Hospital. Each case was accompanied by comprehensive X-ray documentation and confirmed for digit malformations by a consensus of two orthopedic surgeons, with classification according to the Temtamy\u0026ndash;McKusick schema. Furthermore, cognitive assessments, along with evaluations of craniofacial morphology, stature, foot, and finger lengths, were systematically conducted for all subjects to meticulously exclude concurrent congenital anomalies, thereby ensuring a homogenous cohort comprising solely non-syndromic pre-axial polydactylies. Detailed personal and familial medical histories were meticulously recorded, and blood specimens were procured from both probands and their pedigrees based on their individual genetic profiles.\u003c/p\u003e\n \u003cp\u003eThis study was approved by the Institutional Review Board of West China Hospital of Sichuan University with the reference number 2022 Review NO.1623. Written informed consent was obtained from all individual participants recruited in the study or their legal guardians\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003eMutations reported\u003c/h2\u003e\n \u003cp\u003eBy reviewing existing literature, we conducted a comprehensive summary of reported point mutations within the \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e regions associated with PPD1 and PPD2 (Table \u003cspan\u003e1\u003c/span\u003e). Additionally, we summarized information on copy number variations encompassing the \u003cem\u003eZRS\u003c/em\u003e fragment that have been linked to PPD 1 and PPD2 (Table \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Previously reported \u003cem\u003eZRS\u003c/em\u003e duplications causing limb development defects.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"1077\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eLohan et al. [2014]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eLohan et al. [2014]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eLohan et al. [2014]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eWieczorek et al. [2009]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eWu et al. [2009]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eDai et al. [2013]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eSun et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eLohan et al. [2014]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eXu et al. [2019]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eXu et al. [2020]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003eMutation ref\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e16kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e47kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e75kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e89 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e97 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e115 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e160kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e179 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e180 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e182kb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003eHg19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,578,108-156,594,751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,563,856-156,610,632\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,570,780-156,646,750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,572,751-156,661,877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,547,469-156,644,074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,505,616-156,620,919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,539,605-156,699,998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,491,887-156,671,016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,554,005-156,736,751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003eChr7: 156,554,005-156,736,751\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003ePreaxial polydactyly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003eTriphalangeal thumb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003ePartial or complete syndactyly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.17516218721038%\"\u003e\n \u003cp\u003eFeet affected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.082483781278961%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"1074\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eLohan et al. [2014]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eSun et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eWieczorek et al. [2009]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eZlotina et al. [2020]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eSun et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eSun et al. [2008];\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eSun et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eSun et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eKlopocki et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eLiu et al. [2008]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003eMutation ref\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e255 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e265 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e293 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e300 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e334 kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e378kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e437kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e459kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e589kb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e0.29Mb\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003eHg19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,437,229-156,692,706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,354,085-156,619,399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,368,541-156,661,877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,385,810-156,684,811\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,354,085-156,687,613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,241,020-156,619,399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,241,020-156,677,759\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,241,020-156,699,998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,143,386-156,732,204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003eChr7: 156,484,201-156,772,643\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003ePreaxial polydactyly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003eTriphalangeal thumb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003ePartial or complete syndactyly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.260991580916745%\"\u003e\n \u003cp\u003eFeet affected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.073900841908326%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eSanger Sequencing\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003cp\u003eGenomic DNA was isolated from whole blood using an TIANamp Blood DNA Kit (DP348-03, TIANGEN, Beijing, China) following the manufacturer\u0026rsquo;s instructions. Subsequently, primers were designed to amplify the sequences of the ZRS and preZRS genes using polymerase chain reaction (PCR), followed by Sanger sequencing. The primer sequences are listed in Table \u003cspan\u003e3\u003c/span\u003e. PCR was performed in a total volume of 25 \u0026micro;L per reaction, containing 12.5 \u0026micro;L 2\u0026times;Hieff\u0026ensp;PCR Master Mix (10102ES03, YEASEN, Wuhan, China), 1 \u0026micro;L genomic DNA (~\u0026thinsp;50 ng), and 0.5 \u0026micro;l of each primer (5 \u0026micro;M). Reactions were carried out in a T100 Thermal Cycler (Bio-Rad, Hercules, the USA) with the following conditions: 95℃ for 3 min followed by 35 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 60 s.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1722857835.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e. Clinical feature in polydactylies of this study\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.27710843373494%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003ePolydactyly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.19277108433735%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.85542168674699%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eUpper Limb (N)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.85542168674699%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eLower Limb (N)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.819277108433734%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eFamily history\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eRight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eLeft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eBoth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eRight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eLeft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eBoth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.27710843373494%\" valign=\"top\"\u003e\n \u003cp\u003ePreaxial-I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.19277108433735%\" valign=\"top\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.819277108433734%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.27710843373494%\" valign=\"top\"\u003e\n \u003cp\u003ePreaxial-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.19277108433735%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.819277108433734%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.27710843373494%\" valign=\"top\"\u003e\n \u003cp\u003eTOTAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.19277108433735%\" valign=\"top\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.819277108433734%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e. The bioinformatic prediction of the variants/mutations identified\u003c/p\u003e\n \u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003egene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eChange\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eVariant ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eFrequency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003econservation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eClinVar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003eZRS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e3C\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003echr7: 156584567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003ers10254391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003ecommon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003ebenign\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e93G\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003echr7:156,584,477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e106G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003echr7:156,584,464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003ers1354594327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003erare\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e278G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003echr:156,584,292\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003ers1395888355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003erare\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.285714285714286%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003epreZRS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e409 A\u0026gt;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003echr: 156,585,378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e603G\u0026gt;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003echr: 156,585,184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003ers6949624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003ecommon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003ebenign\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e. Summary of the RCN of \u003cem\u003eZRS\u003c/em\u003e region in this cohort using 1.3 as the cut-off value\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRCN*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026ge;1.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;1.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e68 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e34 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e*RCN, Relative copy number\u003c/p\u003e\u0026nbsp;\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003eReal-time quantitative PCR (qPCR)\u003c/h2\u003e\n \u003cp\u003eBased on the \u0026Delta;\u0026Delta;CT method, qPCR was employed to confirm the duplication of fragments containing the \u003cem\u003eZRS\u003c/em\u003e sequence. Primers were designed using the NCBI Primer-BLAST tool (\u003cspan\u003e\u003cspan\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/\u003c/span\u003e\u003c/span\u003e) with reference to previous reports by Sun et al.(Sun et al. \u003cspan\u003e2008\u003c/span\u003e) (Table \u003cspan\u003e3\u003c/span\u003e). Each qPCR reaction had a total volume of 10 \u0026micro;L, consisting of 5 \u0026micro;L 2X Hieff qPCR SYBR Green Master Mix (11201ES08, YEASEN, Wuhan, China), 1 \u0026micro;L genomic DNA (~\u0026thinsp;10 ng), and 0.2 \u0026micro;L of each primer (2 \u0026micro;M), with triplicates for each sample. Reactions were performed on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, USA), with the following cycling conditions: 95℃ for 3 min and 40 cycles of 95℃ for 15 s, followed by 60℃ for 30 s. After the final cycle, melt curve analysis was conducted to confirm amplification specificity. The experiments were replicated three times. A cut-off Relative Copy Number (RCN) of 1.3 was used to define duplication(Sun et al. \u003cspan\u003e2008\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eBioinformatic Analysis\u003c/h3\u003e\n\u003cp\u003eDNA variants were characterized and compared with those reported in public single nucleotide polymorphism databases (\u003cspan\u003e\u003cspan\u003ehttps://www.ncbi.nlm.nih.gov/snp\u003c/span\u003e\u003c/span\u003e), UCSC (\u003cspan\u003e\u003cspan\u003ehttp://genome.ucsc.edu/\u003c/span\u003e\u003c/span\u003e) and the 1000 Genomes dataset (\u003cspan\u003e\u003cspan\u003ehttp://browser.1000genomes.org/index.html\u003c/span\u003e\u003c/span\u003e) were used for identification of SNP and conservation of noncoding sequences.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eClinical features\u003c/h2\u003e\n \u003cp\u003eA comprehensive clinical examination was conducted on the 102 patients included in this study; the clinical features are summarized in Table\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e. All the patients exhibited normal intelligence, facial features, body morphology, and hearing. Among the patients, 96 (94.12%) were diagnosed with PPD1 and 6 (5.88%) with PPD2. Bilateral hand involvement was observed in 12 cases (11.76%), while 90 cases (88.24%) presented with polydactyly in only one hand (right: 54 cases, 52.94%; left: 36 cases, 35.29%). Additionally, three patients (2.94%) exhibited bilateral foot involvement. These instances of polydactyly were predominantly sporadic, with only two cases showing familial inheritance. The study also included other affected individuals from these families.\u003c/p\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003ePoint mutations\u003c/h2\u003e\n \u003cp\u003eSanger sequencing of \u003cem\u003epreZRS\u003c/em\u003e and \u003cem\u003eZRS\u003c/em\u003e in the 102 patients identified six point mutations: 3C\u0026thinsp;\u0026gt;\u0026thinsp;G (chr7:156584567), 93G\u0026thinsp;\u0026gt;\u0026thinsp;T (chr7:156584477), 106G\u0026thinsp;\u0026gt;\u0026thinsp;A (chr7:156584464), 278G\u0026thinsp;\u0026gt;\u0026thinsp;A (chr7:156584292), 409A\u0026thinsp;\u0026gt;\u0026thinsp;C (chr7:156585378), and 603G\u0026thinsp;\u0026gt;\u0026thinsp;C (chr7:156585184) (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). Of these identified variants, 3C\u0026thinsp;\u0026gt;\u0026thinsp;G (rs10254391) and 603G\u0026thinsp;\u0026gt;\u0026thinsp;C (rs6949624) were common genetic polymorphisms observed in the multiple cases, with 46 (45.10%) heterozygous for 3C\u0026thinsp;\u0026gt;\u0026thinsp;G and 38 (37.25%) homozygous for this variant. Similarly, for the 603G\u0026thinsp;\u0026gt;\u0026thinsp;C variant, 43 cases (42.16%) were heterozygous and 47 (46.08%) were homozygous. Both variants are classified as benign according to ClinVar. Mutations, while 106G\u0026thinsp;\u0026gt;\u0026thinsp;A (rs1354594327) and 278G\u0026thinsp;\u0026gt;\u0026thinsp;A (rs1395888355) are classified as rare SNPs, and 93G\u0026thinsp;\u0026gt;\u0026thinsp;T and 409A\u0026thinsp;\u0026gt;\u0026thinsp;C have no prior records. Conservation analysis revealed that the nucleotides at positions 156584477G, 156584464G, and 156585184G were not evolutionarily conserved, whereas 156584567C, 156584292G, and 156585378A were conserved (Table\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eDuplication mutations\u003c/h2\u003e\n \u003cp\u003eOnly four patients were found to have potentially pathogenic point mutations, representing a relatively low frequency. To investigate whether \u003cem\u003eZRS\u003c/em\u003e duplications caused PPD in these 102 patients, primers targeting the region 738-858bp (chr7: 156585427\u0026thinsp;\u0026minus;\u0026thinsp;156585307) upstream of \u003cem\u003eZRS\u003c/em\u003e were designed and the RCNs were determined. The qPCR results showed that 68 of the 102 patients had RCN values greater than 1.3 (range: 1.31\u0026ndash;2.88), defining them as duplication-positive (66.67%; Tables\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e). Additionally, all the patients with non-polymorphic SNP point mutations detected by Sanger sequencing were also duplication-positive (93G\u0026thinsp;\u0026gt;\u0026thinsp;T: 1.31; 106G\u0026thinsp;\u0026gt;\u0026thinsp;A: 1.79; 278G\u0026thinsp;\u0026gt;\u0026thinsp;A: 1.92; 409A\u0026thinsp;\u0026gt;\u0026thinsp;C: 1.74).\u003c/p\u003e\n\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLimb development disorders (LDDs) are the second most common congenital developmental defects after congenital heart disease(Dolk et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). LDDs have thus been a major focus of research and serve as classical models for the study of embryonic morphogenesis and signal regulation(Zhang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although LDDs are reported to be caused by a combination of genetic and environmental factors(Alexander et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), many of the pathogenic mutations responsible for these malformations remain unidentified(Malik \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and even when specific genes have been found to be causative, only a small number of cases exhibit mutations in these genes(Lopez-Rios \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For instance, this study focused on PPD, for which previous research has identified various point mutations within the \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e sequences as causative for PPD1 and PPD2(Potuijt et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Potuijt et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, intensive screening of affected populations has revealed that only a few cases carry these point mutations. This extremely low detection rate significantly impacts patient diagnosis and treatment(Furniss et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eZRS\u003c/em\u003e gene is a well-studied limb-specific enhancer that regulates \u003cem\u003eSHH\u003c/em\u003e, a morphogen associated with the ZPA, and which plays a crucial role in the anterior-posterior axis development of the limb buds(Williamson et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). Consequently, mutations in \u003cem\u003eZRS\u003c/em\u003e lead to anterior-posterior limb abnormalities, manifesting as incorrect polarity from the thumb to the fifth digit, affecting both the digit number and pattern(Lettice et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tickle and Towers \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Point mutations in \u003cem\u003eZRS\u003c/em\u003e have been confirmed to be associated with PPD1(Malik \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, microduplications involving the \u003cem\u003eZRS\u003c/em\u003e region have been implicated in conditions such as TPT, Haas type syndactyly, and Laurin-Sandrow syndrome(Lohan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wieczorek et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, research by Sagai et al. (Sagai et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) demonstrated that homozygous deletions of \u003cem\u003eZRS\u003c/em\u003e resulted in truncation of the distal limbs in mice. Recent studies have also highlighted the significance of the \u003cem\u003epreZRS\u003c/em\u003e region, a 632 bp region located 585 bp upstream of \u003cem\u003eZRS\u003c/em\u003e(Park et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Xiang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Point mutations in \u003cem\u003epreZRS\u003c/em\u003e have been identified as causative for both PPD1 and PPD2, indicating a shared pathogenic mechanism between these phenotypes(Potuijt et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mutations in \u003cem\u003epreZRS\u003c/em\u003e have similarly been shown to disrupt the regulation of \u003cem\u003eSHH\u003c/em\u003e expression, leading to defective anterior-posterior limb patterning(Potuijt et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth PPD1 and PPD2 are caused by mutations in \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e and exhibit similar phenotypes, namely, preaxial abnormalities. Limb malformations are characterized by reduced penetrance and variable expressivity, resulting in different phenotypes within the same family due to differential expression(Duan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, it is hypothesized that PPD1 and PPD2 may represent phenotypes of the same underlying pathogenic mechanism with different expressivity.\u003c/p\u003e \u003cp\u003eThe present study included primarily sporadic cases of unilateral PPD1, with few accompanying foot deformities. These findings align with the known distribution patterns of PPD1 and PPD2. The results indicated a slightly higher incidence of right-hand deformities compared to left-hand, although the \u003cem\u003eSHH\u003c/em\u003e-ZPA-based pathogenic mechanism does not predispose to one side, suggesting that this might be a coincidental observation.\u003c/p\u003e \u003cp\u003eIn this study, four novel potentially pathogenic point mutations were identified. Among these, both the 278G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation within \u003cem\u003eZRS\u003c/em\u003e and the 409A\u0026thinsp;\u0026gt;\u0026thinsp;C mutation within \u003cem\u003epreZRS\u003c/em\u003e show strong evolutionary conservation. Furthermore, the 278G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation is a low-frequency SNP, indicating a strong likelihood of pathogenicity. However, a significant number of cases showed \u003cem\u003eZRS\u003c/em\u003e duplications and elevated RCN values were observed in all four cases with the novel point mutations. It is thus hypothesized that point mutations in both \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e and \u003cem\u003eZRS\u003c/em\u003e duplications contribute to a common pathogenic mechanism leading to PPD1 and PPD2. This suggests that \u003cem\u003eZRS\u003c/em\u003e duplications are pathogenic not only for PPD2 but also for PPD1. Therefore, when investigating the causes of PPD1 and PPD2, it is recommended to screen for point mutations in both \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e, as well as for \u003cem\u003eZRS\u003c/em\u003e duplications. However, no pathogenic cause was identified in 34 cases in this study, indicating that there are still other unknown etiologies awaiting discovery. For example, in 2021, Jin et al. (Jin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) identified two novel variants in \u003cem\u003eGLIS1\u003c/em\u003e in PPD1 patients, linking \u003cem\u003eGLIS1\u003c/em\u003e to PPD1.\u003c/p\u003e \u003cp\u003eThis study has some limitations, potentially due to potential selection bias as patient enrolment was confined to PPD1 and PPD2 cases. Additionally, the detection of \u003cem\u003eZRS\u003c/em\u003e duplications was performed using qPCR, which is a relative, rather than an absolute, quantification method, potentially leading to less precise results. Overall, the findings not only expand the spectrum of mutations associated with non-syndromic polydactyly but also suggest that similar phenotypes caused by the same gene may share common pathogenic mechanisms. The incorporation of various mutations in genetic screening tests can effectively enhance the detection rate of pathogenic mutations and also contribute to the cost-effectiveness of genetic testing for LDDs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Xiaoli Wang from Shanxi Provincial Children\u0026apos;s Hospital for providing samples used in this study. We also extend our gratitude to the pediatric surgery nursing team at West China Hospital of Sichuan University for their generous assistance in sample collection. Additionally, we appreciate the support provided by the Stem Cell Biology Laboratory and other members of the Laboratory of Pediatric Surgery, West China Hospital, Sichuan University for their generous help in methodology and reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e: Jing Chen and Bin Wang conceived the study and designed the experiments. Siyu Pu conducted the molecular experiments and wrote the manuscript. Jing Chen, Siyu Pu, and Zhibo Wang contributed to data interpretation. Zhibo Wang, Bin Wang, Xueyang Tang, Xiaodong Yang, Jun Jiang, Daoxi Wang, and Yifan Deng performed the clinical assessments and collected the samples. Bo Xiang, Jiayin Yang, and Jing Chen supervised the study. Jing Chen provided critical revisions. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by National Key R\u0026amp;D Program of China (2022YFC2703700, 2022YFC2703704), National Natural Science Foundation of China (32170813), Science and Technology Department of Sichuan (2024NSFSC0651), and 1\u0026middot;3\u0026middot;5 project for disciplines of excellence\u0026ndash;Clinical Research Fund, West China Hospital, Sichuan University (2024HXFH035 \u0026amp; ZYGD23026).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests:\u0026nbsp;\u003c/strong\u003eThe authors have no conflicts of interests or financial disclosures that are relevant to the research or information in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e The authors affirm that human research participants provided informed consent for publication of the images in Figure 1b.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlexander PG, Clark KL, Tuan RS (2016) Prenatal exposure to environmental factors and congenital limb defects. Birth Defects Res C Embryo Today 108: 243-273. doi: 10.1002/bdrc.21140\u003c/li\u003e\n\u003cli\u003eBlum AG, van Holsbeeck MT, Bianchi S (2021) Thumb Injuries and Instabilities. Part 1: Anatomy, Kinesiology, and Imaging Techniques of the Thumb. Semin Musculoskelet Radiol 25: 346-354. doi: 10.1055/s-0041-1730397\u003c/li\u003e\n\u003cli\u003eDolk H, Loane M, Garne E (2010) The prevalence of congenital anomalies in Europe. 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American Journal of Medical Genetics Part A 182. doi: 10.1002/ajmg.a.61757\u003c/li\u003e\n\u003cli\u003eZhang B, He P, Lawrence JEG, Wang S, Tuck E, Williams BA, Roberts K, Kleshchevnikov V, Mamanova L, Bolt L, Polanski K, Li T, Elmentaite R, Fasouli ES, Prete M, He X, Yayon N, Fu Y, Yang H, Liang C, Zhang H, Blain R, Chedotal A, FitzPatrick DR, Firth H, Dean A, Bayraktar OA, Marioni JC, Barker RA, Storer MA, Wold BJ, Zhang H, Teichmann SA (2023) A human embryonic limb cell atlas resolved in space and time. Nature. doi: 10.1038/s41586-023-06806-x\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Preaxial Polydactyly, SHH, ZRS, preZRS","lastPublishedDoi":"10.21203/rs.3.rs-4718373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4718373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003ePreaxial polydactyly (PPD) is a congenital limb malformation, previously reported to be caused primarily by mutations in the \u003cem\u003eZRS\u003c/em\u003e and upstream \u003cem\u003epreZRS\u003c/em\u003e regions. This study investigated genetic variations associated with PPD, focusing on point mutations and copy number variations (CNVs) in the \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e regions.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eComprehensive genetic analyses were conducted on 102 patients with PPD, including detailed clinical examinations and Sanger sequencing of the \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e regions. Additionally, real-time quantitative PCR (qPCR) was used to detect CNVs in the \u003cem\u003eZRS\u003c/em\u003e region. The evolutionary conservation and population frequencies of identified mutations were also evaluated.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eSix point mutations were identified, including four novel mutations with potential pathogenicity, namely, 93G\u0026thinsp;\u0026gt;\u0026thinsp;T (chr7:156584477), 106G\u0026thinsp;\u0026gt;\u0026thinsp;A (chr7:156584464), 278G\u0026thinsp;\u0026gt;\u0026thinsp;A (chr7:156584292), and 409A\u0026thinsp;\u0026gt;\u0026thinsp;C (chr7:156585378). Additionally, qPCR analysis revealed that 66.67% of patients exhibited \u003cem\u003eZRS\u003c/em\u003e duplications. Notably, these duplications were also present in cases with newly identified potential pathogenic point mutations. These findings suggest the possible interaction of point mutations in \u003cem\u003eZRS\u003c/em\u003e and \u003cem\u003epreZRS\u003c/em\u003e through a common pathogenic mechanism, leading jointly to PPD.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eThe findings expand the mutation spectrum associated with non-syndromic polydactyly and highlight that, despite different classifications, anterior polydactyly caused by mutations in \u003cem\u003eZRS\u003c/em\u003e and nearby regions may share common pathogenic mechanisms. The incorporation of various mutation types in genetic screening can effectively enhance the rate of pathogenic mutation detection and contribute to the cost-effectiveness of genetic testing for limb developmental defects, thereby promoting healthy births.\u003c/p\u003e","manuscriptTitle":"Genetic Analysis of Preaxial Polydactyly: Identification of Novel Mutations and the Role of ZRS Duplications in a Chinese Cohort of 102 cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-11 12:03:15","doi":"10.21203/rs.3.rs-4718373/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-17T10:57:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-31T12:05:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T13:31:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211881635094105604553782937141078932018","date":"2024-07-14T18:13:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234521499471078409685900946786036251221","date":"2024-07-12T10:57:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250088620933922319620702385111820581650","date":"2024-07-12T10:48:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-12T09:52:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T15:09:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T15:06:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Genetics","date":"2024-07-10T13:35:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a8d09377-fec7-4b70-86fb-0d9c95cb2207","owner":[],"postedDate":"August 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:12:09+00:00","versionOfRecord":{"articleIdentity":"rs-4718373","link":"https://doi.org/10.1007/s00439-024-02709-7","journal":{"identity":"human-genetics","isVorOnly":false,"title":"Human Genetics"},"publishedOn":"2024-10-24 15:58:04","publishedOnDateReadable":"October 24th, 2024"},"versionCreatedAt":"2024-08-11 12:03:15","video":"","vorDoi":"10.1007/s00439-024-02709-7","vorDoiUrl":"https://doi.org/10.1007/s00439-024-02709-7","workflowStages":[]},"version":"v1","identity":"rs-4718373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4718373","identity":"rs-4718373","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}