ZPA Regulatory Sequence Variants in Chinese Patients With Preaxial Polydactyly: Genetic and Clinical Characteristics

Abstract

Preaxial polydactyly (PPD) is a common congenital abnormality with an incidence of 0.8–1.4% in Asians, characterized by the presence of extra digit(s) on the preaxial side of the hand or foot. PPD is genetically classified into four subtypes, PPD type I–IV. Variants in six genes/loci [including GLI family zinc finger 3 (GLI3), ZPA regulatory sequence (ZRS), and pre-ZRS region] have been identified in PPD cases. Among these loci, ZRS is, perhaps, the most special and well known, but most articles only reported one or a few cases. There is a lack of reports on the ZRS-variant frequency in patients with PPD. In this study, we recruited 167 sporadic or familial cases (including 154 sporadic patients and 13 families) with PPD from Central-South China and identified four ZRS variants in four patients (2.40%, 4/167), including two novel variants (ZRS131A > T/chr7:g.156584439A > T and ZRS474C > G/chr7:g.156584096C > G) and two known variants (ZRS428T > A/chr7:g.156584142T > A and ZRS619C > T/chr7:g.156583951C > T). ZRS131A > T and ZRS428T > A were detected in PPD I cases and ZRS474C > G and ZRS619C > T combinedly acted to cause PPD II. The detectable rate of ZRS variants in PPD I was 1.60% (2/125), while PPD II was significantly higher (9.52%, 2/21). Three bilateral PPD cases harbored ZRS variants (13.64%, 3/22), suggesting that bilateral PPD was more possibly caused by genetic etiologies. This study identified two novel ZRS variants, further confirmed the association between ZRS and PPD I and reported a rare PPD II case resulted from the compound heterozygote of ZRS. This investigation preliminarily evaluated a ZRS variants rate in patients with PPD and described the general picture of PPD in Central-South China.

Introduction

Preaxial polydactyly (PPD) is a common congenital abnormality with an incidence of 0.8–1.4% in Asians, characterized by the presence of extra digit(s) on the preaxial side of the hand or foot (1). Severity varies from mere broadening of the distal phalanx with slight bifurcation at the tip to full duplication of the thumb, including the metacarpals (2). PPD is genetically classified into four subtypes, PPD type I–IV (Table 1) (3). PPD I (OMIM 174400) indicates the duplication of one or more of the skeletal components of biphalangeal thumbs, which is the most common subtype in many populations (2). PPD II (OMIM 174500) refers to isolated triphalangeal thumbs or the thumb duplication with triphalangeal components (4). PPD III (174600) is also known as index finger polydactyly. Thumbs of PPD III cases are replaced by one or two index fingers (5). PPD IV (174700) is polysyndactyly of the thumb (6).

Currently, only six genes/loci [GLI1, GLI3, serine/threonine kinase like domain containing 1 (STKLD1), ZPA regulatory sequence (ZRS), pre-ZRS region, and a deletion of 240 kb from the sonic hedgehog signaling molecule (SHH) promoter] have been identified in isolated PPD cases and ZRS is, perhaps, the most special and well known (7–12). ZRS, the zone of polarizing activity (ZPA) (located in the posterior region of the limb bud) regulatory sequence, is a limb-specific enhancer of SHH, which is located nearly 1 Mb from SHH and within intron 5 of Limb development membrane protein 1 (LMBR1) (4). ZRS can promote the expression of SHH in ZPA during the limb development. SHH diffuses from ZPA (posterior mesoderm) to anterior region of limb bud and there is no SHH in anterior region. The graded distribution of SHH determines the finger pattern. ZRS variants would alter the expression of SHH and cause limb deformities. ZRS variants and duplications had been shown to cause PPD I, PPD II, Werner mesomelic syndrome (WMS) (OMIM 188770), and other limb deformities (such as mirror-image polydactyly and radial ray deficiency) (12–16). The correlation between PPD and ZRS is definite, but most articles only reported one or a few cases, especially in PPD II cases. There is a lack of reports on the ZRS-variant frequency in patients with PPD.

In this study, we recruited 167 sporadic or familial cases with PPD from Central-South China. We identified four ZRS variants in four PPD cases (4/167, 2.40%), including two novel variants (ZRS131A > T/chr7:g.156584439A > T and ZRS474C > G/chr7:g.156584096C > G) and two known variants (ZRS428T > A/chr7:g.156584142T > A and ZRS619C > T/chr7:g.156583951C > T). This study preliminarily investigated the ZRS variant rate in patients with PPD living in Central-South China, expanded the spectrum of ZRS variants, furthered our understanding of PPD, and contributed to genetic diagnosis and counseling of patients with PPD.

Materials and Methods

Patients and Subjects

This study was approved by the Review Board of Xiangya Hospital of Central South University. A total of 167 sporadic or familial PPD cases admitted to the Department of Orthopaedics of Xiangya Hospital were recruited. They were all from Central-South China, especially Hunan province. Almost subjects were preschoolers and informed consent forms were obtained from the patients and their guardians. All the subjects and their guardians consented to participate in this study and to publication of the images. Blood was collected from patients and their blood relations.

Deoxyribonucleic Acid Extraction

Peripheral blood samples were collected from patients and their family members to extract genomic DNA by the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, United States).

Variant Screening

The highly conserved 774-bp region of the ZRS (chr7: 156583796-156584569, hg19) was obtained from the National Center for Biotechnology Information (NCBI) database¹ and primers were designed by Integrated DNA Technologies (IDT)² (Table 2) (17). PCR was operated to amplify the target sequences by CFX384 Touch PCR Amplifier (Bio-Rad, Hercules, CA, United States). PCR product sequences were determined using the ABI 3100 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, United States) by Sanger sequencing performed by Boshang Biotechnology Co., Ltd. (Shanghai, China). For patients who were identified ZRS variants, further genetic screening (using PCR and Sanger sequencing) was used to detect whether they harbored pathogenic variants in GLI3 (NM_000168.6, NP_000159.3), GLI1 (NM_005269.3, NP_005260.1), STKLD1 (NM_153710.5, NP_714921.4), or pre-ZRS. Their primer pairs were also designed by IDT (Table 2).

Prediction of Pathogenicity

MutationTaster³ was applied for predicting the pathogenicity of variants. GnomAD⁴ was used to annotate the minimum allele frequency (MAF) of variants. ZRS sequences of species from Evgeny et al. (18) were used to compare the conservation of variant sites (18).

Results

We recruited 167 cases with PPD from Central-South China, including 154 sporadic patients and 13 families, named as PPD001–PPD167 depending on the order of recruitment. Among these cases, almost subjects (154/167, 92.22%) were Han Chinese and 148 patients had isolated PPD (Table 3). Based on PPD subtypes to divide subjects, 125 patients (74.85%) revealed PPD I, 21 patients (12.57%) had PPD II, and only four cases exhibited PPD III (1/167, 0.60%) or PPD IV (3/167, 1.80%). The rest of PPD subjects (17 cases, 10.18%) presented other organ malformations, such as congenital heart disease, radial ray deficiency, and anal atresia. There were 103 male patients (61.68%) and 64 female patients (38.32%). Most subjects were younger than 3 years old. Except 19 cases without clinical details, the overwhelming majority of PPD I/II was unilateral (109/127, 85.83%), in which PPD, on the right hand, accounted for almost two-thirds (72/109, 66.06%; Table 3).

In accordance with the flow diagram (Figure 1), we identified four ZRS variants in four patients (PPD003, PPD029, PPD116, and PPD154; Table 4). The detectable rate of ZRS variants in PPD I was 1.60% (2/125), while PPD II was significantly higher (2/21, 9.52%). Three of these four patients were with bilateral thumbs involvement, occupying 13.64% of bilateral PPD (3/22). None ZRS variant was identified in patients with left PPD, although they were more than one-third total subjects (37.72%, 63/167).

FIGURE 1

PPD029 Family

The proband of PPD029 (III:1) was a 4-year-old girl, who presented bilateral triphalangeal thumbs (Figures 2A–C). She harbored compound heterozygous variants in ZRS (ZRS474C > G/chr7:g.156584096C > G and ZRS619C > T/chr7:g.156583951C > T) without GLI3, GLI1, STKLD1, or pre-ZRS variants (Figure 2D). ZRS474C > G was inherited from her father (II:2) and another variant was from her mother (II:3). Other family members without variants or with only one variant were unaffected.

FIGURE 2

PPD154 Family

The proband of PPD154 (II:1) was a boy with PPD I on the right hand (Figures 2E–G). He was admitted to our hospital for operative treatment at his age of 1.5 years. We identified a de novo variant in ZRS (ZRS131A > T/chr7:g.156584439A > T) in this patient and did not find suspicious variants in GLI3, GLI1, STKLD1, and pre-ZRS (Figure 2H). His parents were unaffected.

PPD003 Family and PPD116 Family

Known ZRS variant (ZRS428T > A/chr7:g.156584142T > A) was identified in two families with PPD II (PDD003) or PPD I (PDD116). Four variants identified in this study were highly evolutionarily conserved and were predicted to be disease-causing by MutationTaster (Figure 2I and Table 4).

Discussion

Polydactyly is the most common limb malformation in China and PPD is over half (data from National Stocktaking Report on Birth Defect Prevention)⁵. PPD I is the most common subtype and PPD III is rarest (2, 19). In this study, 125 cases (74.85%, 125/167) had PPD I and only one patient (1.80%, 1/167) was diagnosed with PPD III. 17 patients with PPD (10.18%, 17/167) had other organ malformations, including congenital heart disease, radial ray deficiency, and anal atresia. These complications were relatively frequent in patients with PPD. Male patients with PPD are approximately twice as many as female (19). In this study, the proportion of male patients was 61.68% (103/167). This study showed that overwhelming majority of PPD I/II were unilateral (85.83%, 109/127), in which PPD, on the right hand, accounted for almost two-thirds (66.06%, 72/109), consistent with previous studies (19, 20).

In this study, we tested ZRS variants in 167 patients with PPD and identified unique variants (MAF ≤ 0.05) in four cases (2.40%, 4/167). The detectable rate of ZRS variants in PPD I was 1.60% (2/125), while PPD II was significantly higher (9.52%, 2/21). Indeed, most known ZRS variants are identified in PPD II cases [data from the human gene mutation database (HGMD)]⁶. In this study, three ZRS variants were associated with bilateral PPD and 13.64% bilateral PPD cases (3/22) harbored ZRS variants, suggesting that bilateral PPD was more possibly caused by genetic etiologies. Compared with that no ZRS variant was detected by Xiang et al. (20) in 82 Chinese patients with PPD I/II or Rao et al. (21) in 72 Chinese patients with PPD, our identification was fortunate (20, 21). For the remaining 163 cases, we planned to applied chromosomal microarray analysis (CMA), whole-exon sequencing (WES), and genome-wide association study (GWAS) to detect their genetic etiologies. Furthermore, environmental factors, such as alcohol, are causes of limb deformities (22).

Of these four variants, ZRS131A > T and ZRS474C > G were novel and ZRS428T > A and ZRS619C > T had been reported in patients with PPD II (4, 15). ZRS428T > A was identified in both the patients with PPD I (PPD116) and PPD II (PPD003), suggesting the variability of ZRS428T > A-related clinical phenotypes. ZRS131A > T was identified in a sporadic case with PPD I (PPD154). Generally, ZRS variants are associated with PPD II and ZRS was first linked with PPD I by Xu et al. (12). Our report may be the second case worldwide, further demonstrating the correlation between ZRS and PPD I.

PPD029 was a rare case. We found that the proband harbored the compound heterozygote of ZRS (ZRS474C > G and ZRS619C > T). Given that Jacob et al. (23) reported ZRS variant carriers with minor anomalies and underlined the importance of accurate clinical examination in mild triphalangeal thumb families, we carefully checked the phenotypes of her family members with one ZRS variant and did not find any limb defects (cannot completely exclude the possibility of an extremely subtle anomaly) (23). We reasoned that PPD in this family was attributed to combinedly acting by these two variants. PPD II is an autosomal dominant disease and our description indicated that PPD II individuals can be affected with a pattern of autosomal recessive inheritance. A previous study indicated that compared with a heterozygous variant in ZRS (ZRS402C > T), the homozygote led to more severe phenotypes, WMS, manifesting the superimposed effect of ZRS variants and our detection demonstrated again this phenomenon (24). ZRS619C > T had been reported by Mohammad et al. (15) in a Saudi Arabian family presented with variable preaxial deformities of the upper limbs including isolated triphalangeal thumb, PPD, preaxial syndactyly, and absent thumb and radius (15). Some family members suffered from renal agenesis and congenital heart disease. The variant (ZRS619C > T) showed obvious phenotypic heterogeneity in the Saudi Arabian family, whereas the variant was unable to alone trigger PPD in PPD029 family. It suggested that the pathogenicity of ZRS variants may be affected by ethnic difference, individual variation, and/or environmental factor.

ZPA regulatory sequence is a limb-specific enhancer of SHH, which induces the expression of SHH within ZPA (25). SHH expends from posterior mesoderm to anterior region of limb buds and lacks within the anterior-proximal. The expression gradient of SHH is crucial in establishing the number and the identity of the digits during anteroposterior patterning of the limb (26). Duplications involved ZRS or gain-of-function variants in ZRS would promote the expression of SHH in ZPA and then trigger the ectopic expression within the anterior region, where proliferation of mesenchymal cells is increased to cause PPD I/II (27). For four ZRS variants identified by us, their biological functions were not clarified and further studies needed to be performed. But, we predicted the pathogenicity of these four ZRS variants and analyzed their conservation. GnomAD showed that these variants were absent from controls or extremely rare. Thus, we highly suspected that these ZRS variants were their genetic etiologies, which should be further investigated.

Conclusion

In summary, we recruited 167 sporadic or familial cases with PPD from Central-South China and identified four ZRS variants (ZRS131A > T/chr7:g.156584439A > T, ZRS428T > A/chr7:g.156584142T > A, ZRS474C > G/chr7:g.156584096C > G, and ZRS619C > T/chr7:g.156583951C > T) in four patients with PPD (2.39%). Our description about epidemiological investigation of PPD helped us to understand the general picture of PPD in Central-South China. Our detection of two novel ZRS variants not only enrich the genetic map of PPD, but also contributed to genetic diagnosis and counseling of patients with PPD. Furthermore, we reported two patients with PPD I harboring ZRS variants further supporting the link between ZRS and PPD I and a PPD II case caused by the compound heterozygote in ZRS contributing to our understanding of PPD II and its genetic mechanism.

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