A Chinese case of CHST3-related skeletal dysplasia and a systematic review

We reported a case with carbohydrate sulfotransferase 3 (CHST3) spondyloepiphyseal dysplasia and made a systematic review of all previously reported cases. A 14.8-year-old boy underwent clinical, radiological, and genetic evaluations. The patients and five age-matched healthy boys accepted high-resolution peripheral quantitative computed tomography evaluation. All CHST3-related skeletal dysplasia cases from PubMed and Embase were collected and summarized. The genotype–phenotype correlation was analyzed. The proband complained of aggravated joint pain and had a compression fracture of L2 during his second decade. Physical examination showed a height Z score of −4.94, short limbs, and restricted movement of the elbows and knees. X-rays showed carpal epiphyseal dysplasia, enlargement of elbow and knee joints, and subluxation of the left hip. Echocardiography showed abnormal cardiac valves. Compared with the norm, his total and trabecular volumetric bone mineral density (BMD), and the microarchitecture of the trabecular bone had trends to be worse at the distal radius and tibia. Two novel missense variants of c.1343T>G and c.761C>G in CHST3 were inherited from his father and mother, respectively. In the systematic review, short stature, limited joint extension, joint pain, and joint dislocation were the most common characteristics of this disorder. Height Z score and the proportion of hearing impairment had no significant differences between the missense and nonmissense mutations groups. Progressive joint pain and movement restriction are the main characteristics of CHST3-related skeletal dysplasia. BMD and bone microarchitecture of this disorder needs further exploration. There is no apparent genotype–phenotype correlation in this disorder.


Introduction
Spondyloepiphyseal dysplasia (SED) with congenital joint dislocations (MIM 143095), also called "spondyloepiphyseal dysplasia, Omani type (SEDO)" or "carbohydrate sulfotransferase 3 (CHST3)-related skeletal dysplasia", is a kind of sulfation disorder [1,2]. It was first reported in 1974 [3], and in 2004, CHST3 was identified as its pathogenic gene [4]. CHST3-related skeletal dysplasia is an extraordinarily rare skeletal disorder without a known incidence or prevalence, but in 2020, Duz et al. reported that there were nearly 50 patients with CHST3-related skeletal dysplasia worldwide [5]. CHST3-related skeletal dysplasia is mainly characterized by prenatal short stature, linear growth retardation, joint dislocation or subluxation, movement restriction of large joints, and skeletal deformities, including kyphosis, scoliosis, slight shortening of the trunk, and genu valgum [2]. Partially affected individuals have cardiac valve dysplasia and hearing impairment [5].
CHST3-related skeletal dysplasia is inherited as an autosomal recessive disorder, which means that both homozygous and compound heterozygous variants in the CHST3 gene can result in the condition [2]. The CHST3 gene is located at chromosome 10q22.1 and it has 3 exons; it is also sometimes called chondroitin 6-sulfotransferase 1 [6]. The CHST3 enzyme consists of 479 amino acids, which can transfer sulfate from 3′-phosphoadenosine-5′-phosphosulfate to position 6 of the N-acetyl galactosamine residue of chondroitin sulfate [4]. The sulfation of chondroitin is crucial for maintaining the development, structure, and function of cartilage and endochondral bone [7]. Several studies have verified that biallelic variants of the CHST3 gene lead to loss of function of the CHST3 enzyme and a sharp reduction of CHST3 enzyme activity, to the point its activity can hardly be detected in urine or fibroblasts collected from patients [4,8,9].
This study reported a SED case with two novel CHST3 compound heterozygous variants, evaluated the affected individual's volumetric bone mineral density (vBMD) and bone microarchitecture by high-resolution peripheral quantitative computed tomography (HR-pQCT), and summarized the clinical, radiological, and genetic characteristics of all known affected cases with CHST3 biallelic variants by a systematic review.

Materials and methods
Subjects A 14.8-year-old boy was referred to our department due to progressive joint pain for 2 years. The birth history, clinical manifestations, family information, and physical examination of the affected individual were recorded in detail. Peripheral blood samples of the affected patient, his parents, and his older sister were collected for genetic tests. The patient and the family members signed informed consent forms for the genetic tests. Five age-matched healthy boys were also included for HR-pQCT evaluation as the control group. This study was performed with the approval of the Ethics Committee of Peking Union Medical College Hospital (JS-1689) and following the Declaration of Helsinki.

Biochemical assays
Fasting blood samples and urine samples were collected to detect biochemical parameters. The blood and urine routine tests, liver function, renal function, serum calcium, serum phosphorus, alkaline phosphatase (ALP), bone turnover markers, including β-C-terminal telopeptide (β-CTX), antinuclear antibody spectrum, and autoantibodies of rheumatoid arthritis and human leucocyte antigen-B27 (HLA-B27) were measured by an autoanalyzer. Hormones of anterior pituitary axes, including growth hormone (GH), insulin-like growth factor-1 (IGF-1), thyroid function, adrenocorticotropic hormone, cortisol, sex hormones, total 25-hydroxy vitamin D (T25OHD) and parathyroid hormone (PTH), were detected by the chemiluminescence method. All of these parameters were measured, and the reference ranges were obtained from the Department of Clinical Laboratory of Peking Union Medical College Hospital.

Genetic tests
Genomic DNA (gDNA) of the patient and family members was extracted from peripheral blood by the QIAamp DNA Mini Kit (51304, QIAGEN, Germany). As a standard procedure, whole-exome sequencing was performed on the gDNA of the patient. For further verification, the gDNA of the patient and the family members was amplified by polymerase chain reaction and Sanger sequencing was performed for CHST3 variants detection (NM 004273.5). The forward and reverse primers for c.761C>G were 5′-ACTCAGTTCATGTTCCGCC G-3′ and 5′-TCTTCTGCGTGGAGTAGATGC-3′, and those for c.1343T>G were 5′-AGAAGGCCCGCGAGATGTA-3′ and 5′-CTTTCACGAGGAGGGGCATAC-3′. The pathogenic ity of two CHST3 variants was graded according to the American College of Medical Genetics and Genomics (ACMG) guidelines [14].

Statistical analysis
All available data were analyzed by using SPSS version 25.0. Data following a normal distribution are displayed as the mean ± standard deviation, and data with an abnormal distribution are depicted as the median (interquartile range, IQR). For parameters evaluated by HR-pQCT, all parameters were compared by the Mann-Whitney U test between the patient and five healthy boys. In the systematic review, proportions of clinical and radiological characteristics in SEDO patients were calculated according to the formula of "affected cases/available total cases". Z scores of heights between different groups were compared by independent-sample t-tests. Bivariate analysis was applied to determine the relationship between the two parameters. Fisher's exact test was performed between two groups with categorical data. A p value less than 0.05 indicated a statistically significant difference.

Clinical case
The 14.8-year-old affected boy was the second child of the nonconsanguineous parents, born at full term, with a birth weight of 2.95 kg and an unknown birth length. Prenatal examination by ultrasound indicated that the lower limbs were short. Since 6 years old, he had difficulty squatting down and was shorter in stature than his peers, and complained chest pain occasionally. At 7.8 years old, he went to the local hospital due to short stature with a height of 108.4 cm (Z score: −3.93). Further evaluation showed that the peak value of GH was 11.51 ng/ml by an insulin-GH stimulation test (reference range, RR: ≥10 ng/ml), the level of IGF-1 was 160 ng/ml (RR: 64~345 ng/ml), no pathogenic variant was found in the fibroblast growth factor receptor 3 gene, and the levels of enzymes related to mucopolysaccharidosis were all within the normal ranges. Therefore, he was diagnosed with "idiopathic short stature" and accepted recombinant-human growth hormone (rhGH) therapy with a dosage of 0.15 IU/kg for 3 years. His height Z score increased from −3.93 to −3.0. At 11 years old, he experienced sudden low back pain, then a "compression fracture of L2" by X-ray was confirmed, and he accepted conservative management. Since approximately 13 years old, he complained of aggravated bone and joint pain in the shoulders, waist, hip, knees, and lower limbs, but no obvious pain in wrists and ankles. Puberty had not started yet, and there was no mental retardation. The growth chart and time points of pain starting at different sites are shown in Fig. 1. His family members were all healthy.
Physical examination at 14.8 years old showed a height of 139.2 cm (Z score: −4.94), a weight of 45 kg, and a head circumference of 55.5 cm ( Fig. 2A, B). There was a grade II soft blowing-like systolic murmur between the second intercostal at the left edge of the sternum. He had a short neck, short limbs, restricted movement and contractures of the elbows and knees, genu valgum, and no obvious hyperextension of the metacarpophalangeal joints. His visual analog score of tenderness from the thoracic to lumbar spinous process reached 6~7 points, and it reached 8 points for thorax compression pain. The vision and audition were normal.
The results of the biochemical tests were as follows: T25OHD was 18.3 ng/ml (RR: ≥30 ng/ml). The antinuclear antibody spectrum, autoantibodies of rheumatoid arthritis, and HLA-B27 were all negative. Other parameters, including hormones of the anterior pituitary axes, calcium, phosphorus, ALP, β-CTX, and PTH, were all within the normal ranges. Images of X-ray and three-dimensional computed tomography during the different periods were shown in Fig. 2C-J, mainly including a compression fracture of L2, scoliosis, epiphyseal dysplasia, joint enlargement, joint subluxation, and arthritis et al. Echocardiography indicated mild aortic valve stenosis with insufficiency and a suspected bicuspid aortic valve. Therefore, the patient was diagnosed with "spondyloepiphyseal dysplasia" clinically.

Parameters evaluated by HR-pQCT
Compared to the patient, the median age of the control group was 15.0 years old (IQR: 14.2~15.7 years old, p > 0.999), and the medians of their height and height Z score were 168.0 cm (IQR: 160.3~172.8 cm, p = 0.333) and  Table 1. Representative three-dimensional images of the radius and tibia from the affected individual and a 15year-old healthy boy are shown in Fig. 3. All parameters had no significant differences between the patient and the control group. But there was a trend that Ct.Ar, Tot.vBMD, Tb.vBMD, Tb.BV/TV, Tb.Th, Ct.Th, and Ct.Po.Dm in the patient were lower than that in the control group both at the radius and tibia.

Genetic results of the proband and family members
Two novel heterozygous variants of the CHST3 gene were identified in the proband by WES, including c.1343T>G (p.M448R) and c.761C>G (p.P254R). Furthermore, Sanger sequencing confirmed that the c.1343T>G variant was inherited from the father and the c.761C>G variant was inherited from the mother. Neither variant was detected in the older sister. The pedigree and the results of Sanger sequencing are shown in Supplementary Fig. 1. According to the ACMG guidelines, these two novel variants were judged as likely pathogenic variants (Supplementary Table  1). According to the typical characteristics and the clear genetic testing result, the proband was diagnosed with SED with congenital joint dislocations.

Systematic review
As of December 31, 2021, a total of 77 affected patients had been reported among 46 families, of whom 70 individuals had a clear genetic diagnosis, including the affected individual in this study and two aborted fetuses. A family history of short stature accounted for 37.5% (9/24). The proportions of consanguineous marriage, suspected endogamous marriage, and nonconsanguineous marriage accounted for 59.3% (16/27), 11.1% (3/27), and 29.6% (8/ 27), respectively. The median age of diagnosis was 10 years old (IQR: 5.8~17.0 years old). A total of 69.6% (32/46) of patients were found to have short limbs during the prenatal examination or joint dislocation after birth. The birth length was 37~46 cm of 27 affected individuals, and the birth weight was 2.1~3.5 kg of 7 affected individuals. The mean Z score of heights was −4.86 ± 1.48 (n = 30). The height Z scores of males and females were comparable, −4.71 ± 1.59 (n = 20) vs. −5.28 ± 1.25 (n = 9), p = 0.335 (Fig. 4A). The Z score of heights in the children was significantly higher than that in the adults, with the mean values of −4.57 ± 1.40 (n = 24) for the children and −6.01 ± 1.30 (n = 6) for the adults, p = 0.030 (Fig. 4B). Further bivariate analysis indicated that the age at evaluation and the height Z scores had a negative correlation (r = −0.55, p = 0.003, n = 27, Spearman analysis, Fig. 4C). The proportions of clinical and radiological characteristics are summarized in Table 2. Short stature, limited joint extension, joint pain, waddling gait, joint dislocation or subluxation, and rhizomelic shortening were the most common clinical manifestations. Hearing impairment was found among 42.9% of patients. As far as we know, apart from our case, no study has reported fractures in affected individuals with CHST3-related skeletal dysplasia. An abnormal vertebral shape, epiphyseal dysplasia, shortening of the femoral neck, joint enlargement, and osteoarthritis were the most common X-ray changes. Cardiac involvement accounted for 84% by echocardiography, mainly affecting the structure and function of the valves, in which mitral/tricuspid regurgitation and aortic/pulmonary valve stenosis were most common, but ventricular septal defect and valve thickening were relatively less common.
Therapeutic data are limited. Only two patients had accepted rhGH therapy, including the patient in this study. The other patient was treated with rhGH for three years but did not acquire the ideal efficacy. Moreover, that patient developed scoliosis. Seven patients accepted orthopedic surgeries many times. In addition, one patient accepted the atlantoaxial fixation surgery due to atlantoaxial instability.
Among 70 patients with pathogenic variants of CHST3, 81.4% had homozygous variants, and 18.6% had compound heterozygous variants. A total of 44 pathogenic CHST3 variants are shown in Fig. 4D and Table 2 of the supplementary materials. Variants of missense, nonsense, and frameshift accounted for 63.6%, 22.7%, and 6.8%, respectively. Deletion and insertion, fragment deletion, and splicing variants all accounted for 2.3%. A total of 95.5% of the variants were located in exon 3, and 90.9% of the variants were located in the sulfotransferase domain.
Then, we explored whether there was a correlation between the genotype and the phenotype of this condition. Height Z score and hearing were chosen as the representative characteristics of the phenotype. CHST3 variants

Discussion
CHST3-related skeletal dysplasia is a type of SED due to a sulfation disorder [30], with no more than 80 cases reported worldwide until December 31, 2021, according to our count. Indeed, variants in the CHST3 gene reduce its enzyme activity sharply [4,8], but it seems not to affect the expression of the CHST3 enzyme, at least in the variant of p.T141M [26]. Since proteoglycans (PGs) consist of a core protein attached to one to over one hundred glycosaminoglycans (GAGs), the normal sulfation process in GAGs is fundamental for PGs playing their roles in the cellular nucleus, cytoplasm, and ECM [30,31]. However, whether CHST3 variants change the downstream signaling pathways of chondrocytes, osteoblasts and osteoclasts remains unknown. Similar to the results of the systematic review in this study, other studies demonstrated that prenatal and postnatal growth retardation, congenital joint dislocation, movement restriction of large joints, and skeletal deformities, including kyphosis, scoliosis, genu valgum, and clubfeet, are the most typical clinical manifestations of patients with CHST3related skeletal dysplasia [2,5]. Even if the clinical characteristics mentioned above are well known, the following manifestations still deserve attention: joint pain, short stature, hearing impairment, and cardiac involvement. First, joint pain often appears around the age of ten and mainly involves the hip and knee joints [26,27]. Similar to osteoarthritis and Kashin-Beck disease, chondrocytes in these two disorders lack CHST3 expression [32,33].
Second, it is inferred that the negative correlation between height Z scores and age at evaluation may be the comprehensive result of aggravation of spinal deformities, flattening of the intervertebral discs, growth retardation of long bones, and aggravation of the lower limb deformities. In 2013, Song et al. showed that CHST3 was a susceptibility gene for lumbar disc degeneration [34], which might provide partial evidence for the conjecture above. Third, there are approximately 43% of patients have a conductive hearing impairment, which may result from ossicular malformation in the middle ear [5]. Finally, CHST3 is highly expressed in the heart [35], so CHST3 deficiency may cause mild abnormalities in cardiac structure, especially the cardiac valves. However, not all reported patients were evaluated with echocardiograms or accepted cardiac function assessment.
In this study, we evaluated the vBMD and bone microarchitecture of a patient with CHST3-skeletal dysplasia for the first time. Due to the limited cases, no statistically significant differences were found among all HR-pQCT A Comparison of the height Z scores between boys and girls. B Comparison of the height Z scores of these patients as children and as adults. C Bivariate analysis of height Z scores and age at evaluation by Spearman analysis. D Summary of all reported CHST3 variants causing CHST3-related skeletal dysplasia. Variants in black, red, blue, orange, green, and purple represent missense, nonsense, frameshift, indel, deletion, and splicing variants, respectively. Indel insertion and deletion; *p < 0.05 parameters between the patient and the control group. However, Tot.vBMD and Tb.vBMD in the patient seemed to be inferior to those in the control group at the distal radius and tibia in general. What's more, as Fig. 3 shows, the microarchitecture of the trabecular bone and Ct.Th at the distal tibia in the patient was poor relative to the controls, but the deterioration of bone microarchitecture at the distal radius in the patient seemed not as severe as that at the distal tibia. This phenomenon may be explained by Wolff's law; that is, mechanical loading remodels bones by strengthening the spongy and cortical bones [36], while the mechanical loading in the lower extremities of the patient was not as heavy as that in the controls due to joint pain, movement restriction and decreased activity capacity. Taking a worse bone microarchitecture and the history of L2 compression fracture of the patient into consideration, clinicians should pay attention to the fracture risk of these patients.
The following clinical characteristics may help with the diagnosis of CHST3-related skeletal dysplasia, including short limbs during the prenatal examination, congenital joint dislocation, and osteoarthritis since the first decade. Due to the prominent clinical and radiological features, it is not difficult to make a clinical diagnosis of SED, but a definite diagnosis still relies on genetic tests. CHST3-related skeletal dysplasia should be differentiated from other types of SED caused by gene mutations of collagen type II alpha 1 chain (COL2A1), Wnt-inducible signaling pathway protein 3 (WISP3), and transport protein particle complex 2 (TRAPPC2) [37][38][39]. Table 3 summarizes the key points for differential diagnosis in common types of SED.
To date, a total of 44 pathogenic variants in CHST3 have been reported, including two novel variants in our study. The percentage of missense variants is the highest, reaching nearly 64%. Similar to previous studies, we did not find genotype-phenotype correlations [5,25]. This may be because over 90% of variants are located in the sulfotransferase domain, and all loss-of-function variants reduce the enzyme activity, so the phenotype is homogeneous among patients with CHST3-related skeletal dysplasia [2,4].
Regardless of the severity of the disorder, there are no effective methods for treatment. The treatment experience with rhGH is limited, but rhGH is not recommended for these patients due to poor efficacy and its aggravation of scoliosis [8]. The height Z score of our patient had improved by approximately 1 standard deviation after rhGH therapy, but he was still short of stature. Most patients accept orthopedic surgeries many times to ameliorate their activity abilities [6,9,17,22,24]. Whether anodyne can help relieve joint pain effectively in these patients is not clear. During follow-up, in addition to activity, joint function, and joint pain, the extraskeletal manifestations deserve attention and evaluation, • No extra-skeletal manifestation SED spondyloepiphyseal dysplasia, SEDO spondyloepiphyseal dysplasia Omani type, also called spondyloepiphyseal dysplasia with congenital joint dislocations or CHST3-related skeletal dysplasia, SEDC spondyloepiphyseal dysplasia congenital, SEDT-PA spondyloepiphyseal dysplasia tarda with progressive arthropathy, SEDT spondyloepiphyseal dysplasia tarda, CHST3 carbohydrate sulfotransferase 3, COL2A1 type II collagen A1, WISP3 Wnt-inducible signaling pathway protein 3, TRAPPC2 transport protein particle complex 2, AR autosomal recessive inheritance, AD autosomal dominant inheritance, XL X-linked recessive inheritance, ECHO echocardiography including hearing examinations and echocardiograms. The prognosis and clinical outcome have not been reported previously, but it is important to pay attention to the psychological condition, quality of life, pain management, and longevity of these patients.
The strengths of this study are as follows: (1) the identification of two novel variants in CHST3 expands the mutation spectrum of the gene; (2) to the best of our knowledge, this study evaluates the bone geometry, vBMD, and bone microarchitecture in patients with CHST3-related skeletal dysplasia for the first time by HR-pQCT; and (3) the clinical, radiological and genetic characteristics of the disorder are reviewed and summarized comprehensively, and the genotype-phenotype correlations are also explored preliminarily. There are also limitations in this study. First, functional studies were not performed to verify the damaging effect of the two novel variants in the CHST3 gene, such as the detection of the enzyme activity. Second, the results evaluated by HR-pQCT of the affected individual in this study cannot represent the bone health condition of all patients with CHST3-related skeletal dysplasia. Although GH is a strong anabolic agent influencing skeletal growth, the influence of rhGH on the vBMD and bone microarchitecture of this patient remained unclear, since there was a 4-year interval between the cessation of rhGH therapy and the evaluation of HR-pQCT. Finally, to a certain extent, the results of the systematic review may have a bias due to the limited data extracted from the literature.
In conclusion, this study reported a CHST3-related skeletal dysplasia case with two novel CHST3 compound heterozygous variants. The affected individual had a history of compression fracture of L2, so clinicians should be made aware of increased fracture risk among these patients. The bone microarchitecture of patients with CHST3-related skeletal dysplasia needs further exploration. By systematic review, short stature, limited joint extension, joint pain, and joint dislocation are the typical characteristics of this disorder, and height Z scores and age at evaluation have a negative correlation. Moreover, missense variants are the most common type in the CHST3 gene. There is no genotype-phenotype correlation in CHST3-related skeletal dysplasia.

Data availability
The raw datasets generated and/or analyzed during this study are not publicly available but are available from the corresponding author on reasonable request.