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

doi:10.21203/rs.3.rs-4718373/v1

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

https://doi.org/10.21203/rs.3.rs-4718373/v1

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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

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

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
Polydactyly	n	Right	Left	Both	Right	Left	Both	Family history
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	-
preZRS	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.

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).

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.

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.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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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

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

Status:

Version 1

Abstract

Background:

Methods:

Results:

Conclusion:

Figures

Introduction

Patients and Method

Patients

Mutations reported

Real-time quantitative PCR (qPCR)

Bioinformatic Analysis

Results

Clinical features

Point mutations

Duplication mutations

Discussion

Declarations

References

Table 1

Additional Declarations

Supplementary Files

Status:

Version 1