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Introduction

Short rib-polydactyly syndrome (SRPS) consists of a group of rare, genetically heterogeneous, autosomal recessive osteochondrodysplasias, which are characterized by short ribs and limbs, hypoplastic thorax, polydactyly and multisystem organ abnormalities (1). An accurate diagnosis of SRPS is difficult to obtain using prenatal ultrasound technology alone, due to overlapping phenotypic features that are characteristic of a large numbers of skeletal dysplasias (2,3). Indeed, four different types of SRPS have been identified, including SRPS1/3 (MIM 613091), SRPS2 (MIM 263520), SRPS4 (MIM 269860) and SRPS5 (MIM 614091) (4). SRPS3 is a perinatal lethal skeletal disorder, with or without polydactyly, which is characterized by a constricted thoracic cage, short ribs, shortened tubular bones and a ‘trident’-like appearance of the acetabular roof.

The present study aimed to further investigate the pathogenicity of two pairs of compound heterozygotes identified in two retrospective samples and search for new molecular etiology in a third SRPS3 fetus. The results revealed novel radiological and histopathological changes observed in the two retrospective samples (5,6). The present study also reported the case of a separate Chinese family, wherein a fetus was diagnosed with skeletal dysplasia. Targeted next-generation sequencing (NGS) panels were used to evaluate 463 known skeletal dysplasia-associated genes in both the parents and fetus. Hematoxylin and eosin (H&E) staining was also conducted. Typical and atypical histopathological features, typical radiological changes and novel dynein cytoplasmic 2 heavy chain 1 gene (DYNC2H1) compound heterozygous variants were identified. The findings of the present study may facilitate the clinical and molecular diagnosis of SRPS3.

Materials and methods

Case presentation

Between January 2015 and December 2018, recruitment of patients and the collection of samples, from two Chinese families, were described in previous reports (5,6). The present study also included another two-generation nonconsanguineous Han Chinese family, focusing on a patient with SRPS3. The mother, a 30-year-old woman with no history of skeletal dysplasia, was referred to the Liaoning Centre for Prenatal Diagnosis (Shenyang, China) at 21 weeks of gestation because prenatal sonography had identified a fetus with short limbs and a narrow thorax (Fig. 1). Further ultrasonography revealed a normal amount of amniotic fluid, a thoracic circumference of 10.6 cm (<5th percentile), a femur length of 1.6 cm (<5th percentile) and a humerus length of 1.9 cm (<5th percentile). The biparietal diameter and head circumference of the fetus measured 4.9 and 18 cm, respectively, which was normal for the gestational age. Abnormalities were not detected in the liver and pancreas, but the kidneys were polycystic. The present study was approved by the ethics committee of Medical Scientific Research and New Technology of Shengjing Hospital of China Medical University (approval no. 2016PS159K; Shenyang, China). Informed consent was obtained for each participant included in the study.

Figure 1. Radiographs of three cases of SRPS3. (A and B) Radiographs of retrospective SRPS3 samples. (C) Radiographs of the newly identified SRPS3 sample. In all three cases, we observed a trident appearance of the acetabulum (as indicated by the red ring); a long, narrow thorax with short ribs; shortened long bones; shortened femurs and remarkable spurs at the metaphysis of the long bones. Case 3 showed obvious congenital bowing of both femurs. SRPS3, short rib-polydactyly syndrome type III.

H&E staining

Sections from each paraffin block of the distal femur growth plates, taken from the previously reported cases, the newly identified case and aborted fetuses without skeletal malformations were processed and stained with H&E according to routine protocols. In brief, the distal femur growth plates were fixed in 4% paraformaldehyde for 24 h at 4°C, followed by decalcification with a decalcifying solution. Subsequently, the samples were washed in tap water for 24 h to clean the tissues, and then dehydrated in serially graded ethanol solutions and embedded in paraffin (Tissue Tek processor and Leica embedder; Leica Microsystems, Inc.). The sections (5 µm) were sagittally sectioned at a thickness of 5 µm, and deparaffinized in xylene, rehydrated in descending concentrations of alcohol, and stained with H&E (Thermo Fisher Scientific, Inc.) using routine protocols. The images were acquired under at ×10 and ×40 magnification using a light microscope (Nikon ECLIPSE Ci; Nikon Corporation).

NGS and Sanger sequencing

Peripheral blood was collected from the parents and amniotic fluid was collected from the fetus. Targeted capturing, NGS and data analysis were conducted as previously described (6). In brief, genomic DNA (case 3 I-1, I-2 and II-1) sequences were captured by a customized capture array (NimbleGen; Roche Applied Science), which was designed to capture all exons and splicing areas of 463 genes known to be associated with genetic skeletal disorders, including SRPS. The library was sequenced on an Illumina HiSeq 2000 (Illumina, Inc.). NGS produced >200 times the average sequencing depth and >98.95% overall coverage. The human reference sequence GRCh37.-hg19 was used to validate the experimental sequencing data. The pathogenicity of variants was annotated by referring to the ClinVar dataset (https://www.ncbi.nlm.nih.gov/clinvar/), Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/) and the Standards and Guidelines for the Interpretation of Sequence Variants of American College of Medical Genetics and Genomics (7). Variants with minor allele frequencies (<0.01) in any of the following databases were selected: gnomAD (https://gnomad.broadinstitute.org/), Exome Aggregation Consortium (https://gnomad.broadinstitute.org/), 1000 Genomes Project (https://www.internationalgenome.org/) and an in-house database. The M-Cap tool (M-CAP version 1.4; http://bejerano.stanford.edu/MCAP/) was used to predict the variant pathogenicity. As an autosomal recessive inheritance model should be applied according to the pedigree chart, variants of compound heterozygotes or homozygotes should be relevant first. The DYNC2H1 variants were amplified by general PCR and sequenced using two DYNC2H1-specific primer pairs, as follows: Primer7883 forward, 5′-GCCAATATATTTATCCAGGATTACC-3′ and Primer7883 reverse, 5′-AAACCAAATAAAGCAAAAGAGA GTG-3′; and Primer6591_6593 forward, 5′-TGAGTTTAAAAATGGTTCTTGAAAAGG-3′ and Primer6591_6593 reverse, 5′-CAAATCATTGTGTTTTGGCAGTTAAG-3′.

Results

Radiographic presentation of SRPS3 cases

Radiographs of all three cases demonstrated similar skeletal characteristics, including the typical phenotypic trident appearance of the acetabulum; a long, narrow thorax with short ribs; shortened long bones; shortened femurs and remarkable spurs at the metaphysis of the long bones (Fig. 1A-C). In addition, case 3 showed obvious congenital bowing of both femurs (Fig. 1C and Table I).

Table I. Clinical features of cases with SRPS3.

Table I.

Clinical features of cases with SRPS3.

	Cases
Clinical features	Case 1	Case 2	Case 3
CS	No	No	No
Week of diagnosis, wg	25	28	21
Diagnosis	SRPS3	SRPS3	SRPS3
Chest circumference, cm	NA	11.2	10.6
Abdominal circumference, cm	19.9	13.8	14.4
Femur length, cm	3.4	2.1	1.6
Humerus length, cm	3	2.1	1.9
Polydactyly	No	No	No
Kidney anomaly	No	No	Polycystic kidney
Liver/pancreas microscopic changes	No	No	No
Trident appearance of acetabulum	Yes	Yes	Yes
Short ribs	Yes	Yes	Yes
Short long bones	Yes	Yes	Yes
Long bones, metaphysis spurs	Yes	Yes	Yes
Congenitally bowed femurs	No	No	Yes
Histopathological changes	Irregularly organized proliferation and hypertrophy zones	Irregularly organized proliferation and hypertrophy zones	NA
Other features	NA	NA	NA

[i] SRPS3, short rib-polydactyly syndrome type III; CS, consanguinity; wg, weeks of gestation; NA, not available.

Cartilage growth plate abnormalities due to DYNC2H1 variants

Histological sections of the distal femur growth plates obtained from the three cases were stained with H&E (Fig. 2). As shown in Fig. 2D, the growth plate of the femur bone from an aborted fetus without skeletal malformations had regular organized zones of proliferation and hypertrophy. However, in case 1, zones of proliferation and hypertrophy could not be visualized; thus, the chondrocytes did not appear to be proliferating. Within the region of osteogenesis, the primary spongiosum showed an abundance of retaining cartilage (Fig. 2A). By contrast, in cases 2 and 3, although proliferation was evident, irregular columnar formations and a lack of normal chondrocytes were observed; indeed, the chondrocytes appeared to be increasingly hypertrophic. Disorganization and increased vacuolization were evident in the region of osteogenesis (Fig. 2B and C).

Figure 2. Photomicrographs of the femur growth plate of three cases and a control. (A) Proliferation and hypertrophy were not visualized in case 1. Primary spongiosum showed an abundance of retaining cartilage (magnification, ×100). Irregular columnar formations and a lack of chondrocytes were observed in cases 2 (B) and 3 (C) (magnification, ×200). Disorganization and increased vacuolization were evident in the region of osteogenesis. (D) Regularly organized zones of proliferation and hypertrophy were seen in the growth plate of a femur bone from an unaffected control fetus (magnification, ×200).

Novel variants identified in SRPS3 case 3

Targeted exon sequencing of 463 known skeletal disorder-related genes was performed in the newly identified proband and parents (case 3, Fig. 3A-3). Together, two compound heterozygous maternal and paternal variants were identified in the DYNC2H1 gene: NM_001080463.1, c.6591_6593delTGG (chr11:103055738-103055740) and NM_001080463.1, c.7883T>C (chr11:103070000), respectively. These results were further confirmed by Sanger sequencing. The two variants were highly conserved upon comparison of the sequences over a range of species (Fig. 3B).

Figure 3. DYNC2H1 variants identified in three short rib-polydactyly syndrome type III families. (A) Pedigree and variants identified in three cases, (A-1) case 1, (A-2) case 2 and (A-3) case 3. (B) Conservation of DYNC2H1 residues. The identified mutant amino acids were highly conserved between different species. DYNC2H1, dynein cytoplasmic 2 heavy chain 1 gene.

Discussion

All three fetuses considered in this report showed prenatal sonographic findings that were consistent with SRPS and all parents agreed to induced labor. Radiography showed similar phenotypes for the three fetuses (Fig. 1). These findings were consistent with previous reports regarding the radiographic presentation of SRPS3 cases (5,6). Although other multisystem anomalies are often associated with SRPS (8), only a polycystic kidney was found in case 3 (Table I).

A number of histopathological changes have been found in the growth plates of patients with SRPS. Such phenomena have been observed in several skeletal dysplasia-associated genes, including intraflagellar transport protein 43 homolog (IFT43), IFT80 and IFT52 (9–11). However, few histopathological changes have been reported in the growth plates of cases involving DYNC2H1 variant-induced SRPS (7). To confirm the involvement of DYNC2H1 variants in endochondral ossification, the histological sections of femur growth plates were stained. Case 1 was atypical, wherein proliferation was absent and an abundance of retaining cartilage was observed in the primary spongiosum (Fig. 2A). Phenotypes similar to those reported in the literature were observed in cases 2 and 3 (Fig. 2B and C).

Variants in DYNC2H1 can inactivate the retrograde ciliary motor, causing skeletal dysplasia (12). A number of the morphological abnormalities observed in human ciliopathies are probably caused by disruption of the hedgehog signaling pathway, including variation in the DYNC2H1 gene (13,14). Although different histopathological changes were observed in the three cases evaluated in the present study, they all showed an abnormal pattern of proliferation and differentiation at the femur growth plates. This may be due to disruption of the hedgehog signaling pathway caused by a DYNC2H1 variant.

One of the novel DYNC2H1 variants found in case 3 (Fig. 3A-3), namely c.7883T>C(p.Leu2628Ser) (chr11:103070000), is located in exon 49 within an ATP binding and hydrolysis domain (AAA_4 domain). The M-Cap tool predicted this variant to be ‘deleterious’. Regardless of this prediction, this variant occurs in the conserved AAA_4 domain, which should affect ATP binding and hydrolysis. In addition, another variant, reported to cause SRPS, has been located within close proximity to c.7883T>C (15).

The other variant identified in this study in case 3, c.6591_6593delTGG(Gly2198del) (chr11:103055738-103055740; Fig. 3A-3), might hinder ATP hydrolysis and thus, the generation of adequate cellular energy for the movement of microtubules. An R2205H substitution close to the novel variant has also been reported to cause SRPS (16). Neither p.Leu2628Ser nor Gly2198del were detected in the control database, indicating that these two variants were pathogenic in nature, rather than polymorphisms.

The present study identified atypical histopathological changes and confirmed the radiological features of three SRPS3 cases. In addition, it successfully diagnosed SRPS3 in a Chinese fetus and identified two novel compound heterozygous DYNC2H1 variants. In conclusion, three pairs of compound heterozygous DYNC2H1 variants were identified as pathogenic. The findings of the present study expand the current understanding of histopathological changes associated with DYNC2H1 variants and provide further confirmation of the radiological features of SRPS3. Collectively, the findings should promote more efficient imaging, as well as histopathological and molecular diagnoses of SRPS3.

Acknowledgements

Not applicable.

Funding

The present study was jointly supported by the Natural Science Foundation of China (grant no. 81701462), the National Key R&D Program of China (grant no. 2018YFC1002900) and the 345 Talent Project.

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to the participants not wanting to share all their sequencing data on a public database except for the pathogenic variants, but are available from the corresponding author on reasonable request.

Authors' contributions

CLX, SQX, XY and YL conceived and designed the experiments. CXL, HKJ, JLL and YL assisted in the recruitment of the patients and acquisition of experimental data. CLX, SQX, XY and HQ performed the experiments. SQX, XY, YL and HQ helped in genetic analysis. CLX, SQX, XY, YL and HQ wrote the manuscript. CLX and YL confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the ethics committee of Medical Scientific Research and New Technology of Shengjing Hospital of China Medical University (approval no. 2016PS159K; Shenyang, China). Informed consent was obtained for each participant included in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References