Prenatal diagnosis of a skeletal disorder characterized by rhizomelic shortening of limbs caused by compound heterozygous variants in the PKDCC gene: Case report and literature review

Abstract

Background

The protein kinase domain containing cytoplasmic (PKDCC) gene (OMIM#618821) is associated with bone development. Biallelic variants in the PKDCC gene can cause rhizomelic limb shortening with dysmorphic features.

Case Report

A fetus was found to be rhizomelic limb shortening at 16 weeks of gestation and amniocentesis was performed at 19 weeks of gestation. Genomic DNA extracted from the amniotic fluid was subjected to chromosomal microarray analysis (CMA), and Trio-total whole-exome sequencing (Trio-WES). Sanger sequencing was used to verify the candidate pathogenic variants. CMA was normal, while Trio-WES identified two compound heterozygous variants in the PKDCC gene, namely c.417_c.423delCGGCGCG insTCATGGGCTCAGTACAC(p.G140fs*35) and c.345G>A (p.W115*,379). Then the fetus was aborted and the development of its bone cells were compared with that of a normal fetus of similar gestational age by histopathological examination. Clinical findings of the fetus were shortening humerus and femur, synophrys, much hair on the side face, simian line on the right palm, etc. Histopathological examination showed that the affected fetus had increased proliferative chondrocytes, widened proliferative bands, and delayed bone mineralization.

Conclusions

We reported a prenatal case of rhizomelic shortening of limbs caused by compound heterozygous variants in the PKDCC gene, which emphasized the important role of Trio-WES for diagnosis of skeletal dysplasia in fetuses.

INTRODUCTION

The skeletal development is a carefully-arranged process which includes cell proliferation, maturation, and apoptosis (Crombrugghe et al., 2002). The skeletal dysplasia, which is clinically and genetically heterogeneous, is a group of disorders involving more than 400 pathogenic genes (Mortier et al., 2019). During long bone development, flat proliferative chondrocytes (FPCs) and hypertrophic chondrocytes (HCs), which differentiate from FPCs, secrete collagen for mineralization and support longitudinal bone growth. The protein kinase domain containing cytoplasmic (PKDCC) gene (OMIM#618821), which encodes a putative protein kinase and strongly expressed in branchial arches and limb buds, is essential for skeletal development by appropriate timing of FPC differentiation (Kinoshita et al., 2009). PKDCC^−/−– mutant embryos showed various morphological abnormalities (including short limbs, cleft palate, and shortened intestine), delayed hypertrophy of chondrocytes, and delayed FPC differentiation (Imuta et al., 2009). Although genetic analysis of PKDCC mutant mice revealed the involvement of PKDCC in bone development, its underlying mechanism such as molecular and cellular of the Pkdcc kinase remains unknown and further research is needed.

With the development and application of clinical diagnostic exome sequencing (DES), more genes causing skeletal dysplasia have been discovered. Sajan et al. (2019) carried out patient–parents trio DES on two unrelated patients with similar skeletal abnormalities (rhizomelic shortening of limbs) and dysmorphic features, and discovered a biallelic gene disrupting variants in PKDCC gene. In addition, this was the first report about this gene causing skeletal dysplasia in humans and these two patients established PKDCC as a novel skeletal dysplasia gene.

The detection rate for prenatal diagnosis of the skeletal disorder is 30%. From a literature review on the PubMed database, there are few reports of prenatal diagnosis on the PKDCC gene causing fetal skeletal dysplasia (Yan et al., 2023). In this study, we investigated the potential genetic cause of a fetus with rhizomelic shortening of limbs at 19 weeks of gestation using a sequential detection approach involving karyotyping, chromosome microarray analysis (CMA) and Trio-total whole-exome sequencing (Trio-WES). The results provided reliable evidence for the subsequent genetic counseling to the family.

CASE REPORT Clinical report

The study was approved by the ethics committee of Peking University International Hospital, and informed consent was signed by the recruited couple. A 32-year-old pregnant woman, gravida 1 para 0, was referred to our center in 2021. She is healthy, with no history of chronic diseases such as diabetes and hypertension, no habits such as smoking and drinking, and no exposure to radiation or toxins. Her husband is also healthy, and both she and her husband have no family history of skeletal dysplasia disorders or congenital malformations. She has regular menstruation, and her last menstruation period was October 17, 2021. This was her first pregnancy, and the size of the fetus of the B-ultrasound in the first trimester was consistent with the gestational age. At 12⁺³ weeks of gestation, the ultrasound indicated normal nuchal translucency thickness (0.11 cm), with no obvious abnormalities. Down's syndrome screening was performed at 16⁺³ weeks of gestation. Alpha fetoprotein (AFP), free E3 (uE3), and free β-HCG of maternal serum were detected, combined with maternal age and medical history to calculate the risk using the software (BECKMAN company, Pengcheng prenatal screening analysis software V1.1.0). Her risk value for Down syndrome was 1:4090 (≥1:270 indicates high risk), which showed low risk. At 16⁺³ weeks of gestation, the B-ultrasound showed that there was an omphalocele of 1.6 × 1.3 × 1.3 cm and the femur and humerus were lower than the 5th percentile of similar gestational age. The patient was advised to undergo amniocentesis for genetic testing. Then at 19 weeks of gestation, amniocentesis was conducted and chromosome karyotype analysis, CMA, and Trio-WES of the amniotic fluid were performed. Another B-ultrasound examination was conducted at 22⁺⁴ weeks of gestation, and it was found that the fetus was only equivalent to 20⁺⁶ weeks of gestation, lower than the 10th percentile, with an omphalocele of 2.8 × 1.8 cm, and the femur and humerus less than the 1st percentile. The fetal growth curves of the femur and humerus were shown in Figure 1c,d.

Generate image description

FIGURE 1. The clinical findings in the case. Ultrasonographic indications of the fetus at 22+ gestational weeks showing shortening femur (a) and umbilical hernia (b). The fetal growth curves of the femur and humerus (c, d). Physical appearance of the aborted fetus, which includes synophrys, much hair on the side face (e), simian line on the right palm, etc. (f). X-ray of the fetus showing shortening humerus and femur (g). Pedigree diagram of the family (h).

Genetic testing showed that the fetal karyotyping and CMA were both normal. Two compound heterozygous variants in the PKDCC gene consisting of two variants, namely c.417(exon1)_c.423(exon1)delCGGCGCGinsTCATGGGCTCAGTACAC (p.G140fs*35) and c.345(exon1)G>A (p.W115*,379) were identified by Trio-WES and confirmed by Sanger sequencing which revealed that the father carried the heterozygous c.417(exon1)_c.423(exon1)del variant, while the mother carried c.345(exon1)G>A variant (Figure 2). Both of them were first reported in this study. According to the variant interpretation criteria by ACMG (American College of Medical Genetics and Genomics, 2019), the former one was classified as likely pathogenic, with the evidence of PVS1 + PM2, while the latter was classified as pathogenic, with the evidence of PVS1 + PM2 + PM3.

Generate image description

FIGURE 2. The two variants detected in this case (a and b). Red arrows represent the carrying status of these variants, respectively.

And then genetic counseling was performed. The geneticist's advice was as follows: firstly, the fetus showed fetal growth restriction, with omphalocele and short limb deformity, and the genetic testing indicated a PKDCC compound heterozygous mutation, which was a pathogenic variant and was derived from both parents. The risk of poor fetal prognosis is high and whether to terminate pregnancy or not depends on the couple. Secondly, the recurrent risk would be 25%, so reproductive options such as preimplantation genetic diagnosis (PGD) may be a choice. The couple requested to terminate pregnancy and induction of labor was performed.

After induced labor, a male, weight 680 g, length 32 cm stillborn fetus was discharged. Clinical findings of the fetus were shortening humerus and femur, synophrys, much hair on the side face, simian line on the right palm, etc. X-ray was performed on the aborted fetus, and the lengths of the humerus and femur were measured. It was found that the humerus length was 2.8 cm, at −5.9 SD of the same gestational week, while the femur length was 3.6 cm, at −3.2 SD of the same gestational week (Figure 1).

Histopathological examination found that the fetus with shortening limbs had increased proliferative chondrocytes, widened proliferative bands, and delayed bone mineralization, compared with the control (Figure 3).

Generate image description

FIGURE 3. The histopathological examination of the case (a) and the normal control (b). Histopathological examination showing increased proliferative chondrocytes, widened proliferative bands, and delayed bone mineralization in the fetus with shortening femur and humerus (black arrow).

Genetic analysis

Conventional karyotyping by G-banding was performed on amniotic fluid cells (AFCs) according to standard operation procedures to detect overall chromosomal anomalies. CMA chip with CytoScan 750 K SNP Array (Affymetrix Inc) was conducted according to the manufacturer's manual workflow on DNA extracted from AFCs to investigate genomic copy number variants with clinical significance. Data were collected and analyzed by GeneChip Scanner 3000 with software.

Trio-wes was performed. Protein-coding exome enrichment was performed using xGen Exome Research Panel v2.0(IDT, Iowa, USA) that consists of 429,826 individually synthesized and quality-controlled probes, which targets 39 Mb protein-coding region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled probe space. High-throughput sequencing was performed by MGI DNBSEQ-T7 sequencing instruments (PE150), and not less than 99% of target sequence were sequenced. Raw data were processed by fastp for adapters removing and low-quality reads filtering. The paired-end reads were performed using Burrows-Wheeler Aligner (BWA) to the Ensemble GRCh37/hg19 reference genome. Base quality score recalibration together with SNP and short indel calling was conducted using Genome Analysis Toolkit (GATK). According to the sequence depth and variant quality, SNPs and Indels were screened, and high-quality and reliable variants were obtained. The online system independently developed by Chigene (www.chigene.org) was used to annotate database-based minor allele frequencies (MAFs), and ACMG practice guideline-based pathogenicity of every yielded gene variant, and the system also provide a serial software package for conservative analysis and protein product structure prediction. The databases for MAFs annotation include 1000 genomes, dbSNP, ESP, ExAC, and Chigene in-house MAFs database; Provean, Sift, Polypen2_hdiv, Polypen2_hvar, Mutationtaster, M-Cap, and Revel software packages were used to predict protein product structure variation. As a prioritized pathogenicity annotation to ACMG guideline, OMIM, HGMD (Human Gene Mutation Database) and ClinVar databases were used as conferences of pathogenicity of every variant. To predict functional change of variants on the splicing sites, MaxEntScan, dbscSNV, and GTAG software packages were used instead of product structure prediction software.

Histopathological examination

The cartilage tissue samples from the fetus with rhizomelic shortening of limbs and a normal aborted fetus of similar gestational age as normal control were examined histopathologically. Decalcification and dehydration were performed on the fixed fetal tissue samples, and then paraffin was embedded into wax blocks. Routine pathological sections were performed, 4 μM thick, and then stained with hematoxylin eosin (HE).

The development of bone cells was observed under routine microscopy.

DISCUSSION

Skeletal dysplasia, which includes over 400 disorders, is a heterogeneous group of diseases that affect bone development. Most of the skeletal dysplasia is caused by genetic variants. Due to the development of molecular genetics, our understanding about skeletal dysplasia has increased over time.

The process of bone development is from RPCs (round proliferative chondrocytes) to FPCs and then to HCs. Mutations in the PKDCC gene may result in delayed differentiation of RPCs into FPCs, which then result in delayed formation of HCs. The delayed differentiation of FPCs and HCs, which support the elongation of long bones, lead to shorter mineralization regions of long bones in Pkdcc/P0 mice compared to wild-type mice. Homozygous PKDCC gene knockout mice exhibit severe limb shortening, cleft palate, and craniofacial abnormalities (Imuta et al., 2009).

PKDCC-related skeletal dysplasia is an extremely rare disorder which can be deduced by the small number of the heterogeneous gene disrupting variants in gnomAD. Sajan et al. (2019) reported biallelic PKDCC variants in two patients (one had a nonsense variant p.(Tyr217*) (NM_1 38370 c.651C>A) and the other had a splice donor variant c.639+1G>T) with rhizomelic shortening of limbs and variable dysmorphic features. One patient had skeletal findings, including rhizomelic shortening and milder mesomelic shortening of the upper and lower extremities, short thumbs, bilateral short fifth fingers, hyperextensible fingers, bilateral middle finger clinodactyly, limited range of motion of shoulder joints, chronic joint pain, juvenile idiopathic arthritis, and bilateral patellofemoral joint dislocation. Some mild dysmorphic features such as a prominent forehead, downslanting palpebral fissures, broad nasal bridge, long philtrum and other findings included obesity, left-sided headaches, a café au lait macule on upper right arm, acanthosis nigricans, chronic stage 1 kidney disease, depressed mood, occasional abdominal pain, dizziness, nausea and a left-sided small branchial cleft defect covered with skin without an obvious fistula also existed, without positive cardiovascular, ophthalmological, endocrine, respiratory, hematological, and immune findings. The other patient had rhizomelic short stature most evident in the upper extremity with prominent fingertip pads and a simian crease on the left. Other features included hypotonia, bilateral mild conductive hearing loss, laryngomalacia, polyploid anal mass which had decreased in size and brisk deep tendon reflexes. Craniofacial dysmorphic features comprising macrocephaly, short neck, micrognathia, mild proptosis, depressed nasal bridge, and long smooth philtrum were noted. In 2023, eight biallelic PKDCC variants in a cohort of individuals from seven independent families included six frameshifts, a previously described splice-donor site variant and a likely pathogenic missense variant (Pagnamenta et al., 2023). These eight individuals had consistent rhizomelic shortening, predominantly of the upper limbs, and micrognathia, hypertelorism and hearing loss appeared to be commonly co-occurring features.

Fetal skeletal dysplasia may be suspected because the femur and humerus are found to be short for gestational age or qualitative abnormalities. The accuracy of prenatal testing is critical because it will significantly affect parents' consultation and decision-making on pregnancy management, as well as patients' reproductive choices, including prenatal genetic testing for preimplantation or future pregnancy.

In 2023, Yan et al. (2023) reported the first prenatal case with rhizomelic limb shortening and dysmorphic features caused by compound heterozygous variants of the PKDCC gene, namely, c.346delC(p. Pro117Argfs*113) and c.994G>T(p.Glu332Ter), and neither variant was reported previously. In this case, the dysmorphic features were apparent with rhizomelic shortening of the upper limbs, prominent forehead, and nasal planus. In our study, fetal limb shortening was discovered because the femur and humerus were obviously short for gestational age and then amniocentesis to obtain fetal DNA for trio-WES confirmed rhizomelic shortening of extremities caused by heterozygous variants in the PKDCC gene. One was a frame-shift variant and the other was a nonsense variant, causing early termination of protein translation. Both of them were novelly detected, and this novel finding expanded the variation spectrum of PKDCC gene. and strengthens the link between biallelic inactivation of PKDCC and rhizomelic limb-shortening. The recurrent risk would be 25%, so reproductive options such as PGD may be a choice to the couple. And the outcome of the future pregnancy should be followed up.

In conclusion, we identified novel heterozygous variants in the PKDCC gene causing fetal rhizomelic shortening which would provide guidance for the couple's future pregnancy.

AUTHOR CONTRIBUTIONS

Jing Wang and Huijun Yu drafted the manuscript; Xiaoying Zhang performed the histopathological experiment; Ya Tan and Ying Gu analyzed the WES data; Xiuyun Zhou analyzed the ultrasound data; Zhi Li collected the clinical data; Li Lin designed the study. All authors have agreed to be personally accountable for their own contributions. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS

The authors thank the family in this study.

FUNDING INFORMATION

This work was supported by Ministry of Science and Technology of the People's Republic of China (No. 2018YFC1002201).

CONFLICT OF INTEREST STATEMENT

All authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data presented in this study can be found in online repositories at: https://doi.org/10.6084/m9.figshare.25013273.

ETHICS STATEMENT

The study was approved by the ethics committee of Peking University International Hospital and informed consent was signed by the recruited couple.