A Novel EBP c.452A>G Mutation Identified in a Girl with Conradi–Hünermann–Happle Syndrome Presenting with Hydronephrosis

Introduction

Chondrodysplasia punctata (CDP) is a clinically and genetically heterogeneous disorder characterized by punctiform calcification of the bones with a prevalence of 1–9/1000 000. X-linked dominant CDP (CDPX2, MIM 302960), also referred to as Conradi–Hünermann–Happle(CHH) syndrome,¹ is the most well-characterized form of CDP. CDPX2 arises almost exclusively in females and has been presumed lethal in males, although a few affected males have been reported. There are several characteristics with this syndrome. Firstly, CDPX2 is typically skeletal dysplasia characterized by asymmetrical bone defects including punctiform calcification of the long bones, radiographic epiphyseal stippling, shorten of limbs, scoliosis, short stature, craniofacial abnormalities. Secondly, it manifests with transient severe congenital ichthyosis following linear hypopigmented patches with follicular atrophoderma as well as patches of scarring alopecia. Thirdly, it may show ocular changes including cataracts, microphthalmia or microcornea.^2–4

CDPX2 is caused by mutations in the emopamil-binding protein (EBP) gene, which encodes an integral membrane protein primarily located in the endoplasmic reticulum. This protein functions as a key enzyme in the final steps of the sterol biosynthesis pathway.⁵ Recently, next-generation sequencing applied in family-based and case–control studies has extensively identified specific genes contributing to the risk of complicated hereditary diseases, including dysosteogenesis disorders caused by EBP gene variations.

Herein, we report a 7-year-old girl with short stature, craniofacial defects, extremely severe scoliosis, digital abnormality, malformations with centrums, sectorial cataract in the right eye, ichthyosis and alopecia, and hydronephrosis. Whole exome sequencing (WES) revealed a missense mutation (c.452G>A) in the EBP gene, leading to a genetic diagnosis of CDPX2. Hydronephrosis is not a common feature of CDPX2, and this case represents the first report of the characteristic in China, thereby expanding the phenotype spectrum of the syndrome. Additionally, we reviewed 43 publications, including cases from several EBP reviews, and identified 97 EBP mutations in patients with skin abnormalities, bone defects or cataracts.^1,6–46

Clinical Description

The 7-year-old girl was born via cesarean delivery at 38 weeks of gestation, measuring 45 centimeters in length (−2.7 SD) with short legs, and weighed 3.85 kilograms. Her mother and father are of normal height, 169 cm and 173cm, respectively. Both parents are healthy except that the mother has a history of irregular menstruation and amenorrhea for ten years without pregnancy, and she conceived the baby through ovulation induction therapy. The father has poor sperm motility and hypoplastic lumbar vertebrae. No abnormalities were found during prenatal testing.

She was first referred to our genetics department for evaluation at the age of 4 years. A detailed medical history was collected from birth. There’s no genetic disease history in the family (Figure 1A). Significant developmental delay was observed during her infancy. Ichthyosis persisted from birth to childhood, and hair abnormality included scarring alopecia in patches, sparse eyelashes and coarse, lusterless hair (Figure 1B–D). Cataracts in the right eye, low-set ears and downslanting palpebral fissures were presented in the proband (Figure 1C). She also presented with elbows valgus, flexion of the fingers, and bowleg (Figure 1E and F). Radiographic findings revealed stippling (chondrodysplasia punctata) involving the epiphyses of the long bones and vertebrae (Figure 2A and B). She also had asymmetric shortening of the limbs, primarily affecting the femur and humerus, and severe spinal deformity (Figure 2C and D). Hydronephrosis of the right kidney was onset since her birth (Figure 3A and B).

Figure 1 Clinical features of the 7-year-old proband. (A) Family diagram. (B) Cicatricial alopecia. (C) Sectorial cataract of right eye, sparse eyebrows. (D) Ichthyosis of the leg. (E) Camptodactylia of right hand. (F) Scoliosis and asymmetric shortening of limbs.

Figure 2 Radiological features of spine and limbs of the girl at 2.5 years old. (A) Multiple butterfly like vertebrae and scoliosis. (B) Punctate calcific stippling sternum, ribs. (C) Scoliosis and deformities of ischium and pubis. (D) CT shows neurogenic bladder, and hydronephrosis of right kidney and right ureter.

Figure 3 Voiding cystourethrogram of the girl at 2.5 years old. (A) Hydronephrosis of right kidney and right ureteral dilatation; bladder wall showed a “room beam-like” change. (B) Poor bladder emptying, more contrast agent residue.

Materials and Methods

Whole exome sequence analysis To identify the gene mutation responsible for the patient’s typical phenotype, genomic DNA was extracted from peripheral blood samples from the family after informed consent was obtained. Whole exome sequence analysis was performed. DNA libraries were prepared from the proband and her parents and pooled using a standard protocol. Quantitative PCR was performed to test the quality of the enriched nucleotides. An Agilent Bioanalyzer 2100 was used to determine the fragment size distribution and DNA concentration. Exome capture libraries were prepared using the SureSelect Human All Exon V6 kit (Agilent Technologies, USA). All samples were sequenced on the Illumina HiSeq2500 platform.

After the removal of adapter sequences and the elimination of low-quality reads (where the quality score threshold for raw data was set at Q30 < 85%, and for clean data at Q30 < 90%), the remaining high-quality sequencing reads were mapped to the reference genome (GRCh37/hg19) utilizing the Burrows-Wheeler Aligner (v 0.7). PCR duplicates were subsequently removed using samtools (version 0.1.18). Variant identification and annotation were conducted using the Broad Institute’s GATK (version 4.0) and Annovar software, respectively. The median sequencing depth achieved was 100×, with 98% of the target regions covered at a depth of at least 10×. Variant calls were required to have a minimum of four supporting reads, accounting for over 20% of the total reads at each position; calls failing to meet this threshold were excluded. Variants with a minor allele frequency exceeding 0.5% in public databases (including 1000 Genomes, dbSNP, ExAC, and gnomAD) were filtered out. Prioritization of variants was based on their potential to disrupt protein function, as predicted by tools such as SIFT, Polyphen2, Mutation Taster, Provean, Revel and Human Splicing Finder (version 3.0). Additionally, amino acid conservation across species was evaluated using PhyloP and PhastCons. The correlation between genotype and phenotype played a crucial role in identifying candidate variants, which were further inspected using the Integrative Genomics Viewer for visual validation and confirmed through Sanger sequencing.All the selected variants were then classified as pathogenic, likely pathogenic, VOUS, likely benign or benign according to the American College of Medical Genetics and Genomics (ACMG) guidelines.⁴⁷

Sanger sequencing validation Sanger sequencing was used to validate the variant. Exon 4 of the EBP gene (NM006579.3): c.452A>G(p.Gln151Arg) was amplified using primers F: 5′-GAGCACTAATGGGCTAACCTGTAG-3′ and R: 5’-AGGGATACATCTGTGTCTGTGGAT-3 with an annealing temperature of 60°C. The amplified product length was 275 bp.

Results

WES identified a mutation, c.452A>G (p.Q151R), in the EBP gene, which was confirmed by Sanger sequencing (Figure 4). The mutation was de novo, and the phenotype of the patient was consistent with the disease (ACMG variant evidence: PS2). The mutation was not found in ExAC, gnomAD or 1000G database (ACMG variant evidence: PM2_P); Computational prediction tools unanimously supported a deleterious effect on the gene. Glutamine at position 151 is located in endoplasmic reticulum 2 (ER2) lumen and is conserved across species, including Homo sapiens (human), Macaca Mulatta, Fcatus, Mus musculus (mouse), Takifugu rubripes, and Danio rerio (ACMG variant evidence: PP3); A previously reported amino acid change at position 151, a stop-gain mutation (c.451C>T), has been described in the literature.^9,27 Therefore, the variant c.452A>G (p.Q151R) in the EBP gene was classified as likely pathogenic (PS2, PM2_P, PP3) according to the ACMG guidelines.

Figure 4 DNA analysis of the EBP gene in the proband and her parents by Sanger sequencing.

Discussion

All EBP mutation reports included in this study encompass 141 cases: 86 female, 25 male, 15 female fetuses, and 15 cases with unknown gender (Table 1). The three main malformations associated with CDPX2 are skeletal changes, skin scaling ichthyosis, and ocular abnormalities. Among these cases, epiphyseal stippling was observed in 41.8% (59/141), skin deformities in 78.7% (111/141), and cataracts in 36.9% (52/141). In some cases, skin lesions may not be noticed unless the female has a child with asymmetrical shortening of long bones or ichthyosis-like skin. Digital abnormalities, sparse and lusterless hair, or alopecia are also common features in some patients. In contrast, intra-familial reports show a higher proportion of these manifestations. More than 95% of patients diagnosed with CDPX2 exhibit ichthyosis, while chondrodysplasia punctata, alopecia, and asymmetric shortening of long bones along with short stature are present in 80% of patients. Unilateral or bilateral cataracts are observed in 60% of cases.^8,48 Asymmetrical skeletal deformities and cutaneous lesions are the two primary manifestations of CDPX2. Skeletal changes, including vertebral malformations, epiphyseal stippling, or flocculent calcifications, are notable findings on X-ray.^{11,27,29,35,49}

Table 1 Summary of Major Manifestations in Reported CDPX2

Our proband is a girl affected by a de novo missense variant. Her clinical features are consistent with those of individuals carrying previously reported EBP variants, including multiple butterfly-like vertebrae, severe scoliosis, punctata calcific stippling of the sternum and ribs, deformities of the ischium and pubis on radiographs, cataract in the right eye, sparse eyebrows and eyelashes, and patchy alopecia. Pigmented scales were also observed on her lower legs. Additionally, asymmetric lower limb length and genu varum (bowlegs) were identified in this case.

However, atypical hydronephrosis of the right kidney and right ureter, along with a neurogenic bladder, were observed in this case. This is a rare clinical feature. Among the previous reports, only one female with hydronephrosis has been reported by Herman GE had.⁸ Additionally, thirteen males with EBP mutations have been reported to exhibit renal deformities or abnormalities of the genitourinary system, including urinary incontinence, hypospadias, renal anomalies, cryptorchidism.^9,13,22

In addition, digital abnormalities were reported in 27out of 141 cases in previous studies. The most common features of digital abnormalities include 2–3 toe syndactyly (11/27) and polydactyly or postaxial polydactyly (7/27). However, camptodactyly was observed in this case, a feature not previously reported.

The EBP gene, also known as emopamil binding protein, is localized to the endoplasmic reticulum and functions as a molecular acceptor of emopamil. It was the only gene identified as responsible for CDPX2, as reported by Braverman et al in 1999.³ To date, over 90 mutations in the EBP gene have been reported as causes of CDPX2. Among these, eight mutations were identified in males, four of which were reclassified as male EBP disorder with neurological defects (MEND) syndrome. In the literatures, 37 missense mutations, 24 nonsense mutations, 21 frameshift mutations, and 8 splice variants in the EBP gene were reported (Figure 5).

Figure 5 Mutations in CHH syndrome and MEND syndrome in reports of EBP gene. Ninety-one mutations are concluded. Missense and nonsense mutations are represented using amino acid sequence while deletions, insertions, splicing and initiation mutations showed only with cDNA sequence. EBP mutation of this case was shown with red five-pointed star. *stop codon position in a protein sequence. Abbreviations: N-t, N terminal; TMD, transmembrane domain; ER, endoplasmic reticulum; C-t, C terminal.

Previous studies suggested that EBP frameshift or nonsense mutations, which cause truncated proteins, result in typical CDPX2 with complete clinical features involving skeleton, dermatologic, and ocular manifestations, whereas the clinical severity of missense mutations is variable.² However, in our case, the missense mutation exhibited all typical manifestations, as well as neurogenic bladder, hydronephrosis and camptodactylia. Therefore, the clinical phenotype cannot be predicted solely based on the genotype. WES is particularly useful for diagnosing rare genetic disorders with heterogeneous presentations due to differences in severity or expressivity, as well as the involvement of multiple genes causing similar phenotypes.

To date, the treatment of CDPX2 remains symptom-based. However, it is crucial for the parents to undergo prenatal diagnosis during subsequent pregnancies following a genetic diagnosis.

In conclusion, we identified a novel missense mutation in the EBP gene in a Chinese family, expanding the mutation spectrum of CDPX2. Meanwhile, we reviewed previously reported cases to summarize variations in the EBP gene, which may facilitate clinical applications. Furthermore, we highlight the value of WES in providing genetic diagnoses for rare diseases.