Biallelic variants in CHST3 cause Spondyloepiphyseal dysplasia with joint dislocations in three Pakistani kindreds
BMC Musculoskeletal Disorders volume 23, Article number: 818 (2022)
Skeletal dysplasia is a heterogeneous group of disorders. Spondyloepiphyseal dysplasias comprise one subgroup. Deficiency of carbohydrate sulfotransferase 3 has been reported in a small number of patients with recessively inherited spondyloepiphyseal dysplasia with joint dislocation, short stature and scoliosis. We report here molecular and clinical findings of affected individuals in three consanguineous Pakistani families. Affected individuals in all three families had a uniform phenotype including severe short stature, multiple dislocated joints, progressive scoliosis and facial dysmorphism.
Clinical evaluation was done for three unrelated families. Radiological survey of bones was completed for patients from two of the families. Whole exome sequencing index patients from each family was performed followed by Sanger sequencing for validation of segregation of identified variants in respective families. In-silico analysis for determining pathogenicity of identified variants and conservation was done.
Whole-exome sequencing revealed biallelic variants c.590 T > C;p.(Leu197Pro), c.603C > A;p.(Tyr201Ter) and c.661C > T;p.(Arg221Cys) in CHST3 (NM_004273.5) in the three families with eight, five and two affected individuals, respectively. Contrary to previous reports, affected individuals in none of the families exhibited a hearing loss.
We describe genotypic and phenotypic findings of three unrelated families with spondyloepiphyseal dysplasia. Our study confirms phenotypic variability and adds to the genotypic spectrum of spondyloepiphyseal dysplasia.
Skeletal dysplasias comprise a genetically and clinically heterogeneous group of disorders with more than 460 types involving mutations in more than 350 different genes . Spondyloepiphyseal dysplasia (SED) with congenital joint dislocations, also known as recessive Larsen syndrome (OMIM #143095) is a unique form of skeletal dysplasia characterized by severe short stature, deformed and dislocated joints and progressive kyphosis.
Bone and cartilage are heterogeneous tissues which together make the human skeleton. They are comprised of several types of cells and of the extracellular matrix, which contains various proteoglycans and glycosaminoglycan (GAGs). These GAGs are heavily sulfated and several enzymes are involved in the sulfation of these macromolecules. Sulfation confers a negative charge on GAGs which is important for their functions.
Disease-causing variants in CHST3, encoding chondroitin 6-sulfotransferase 3 (C6ST), were first identified in patients with spondyloepiphyseal dysplasia (SED), Omani type (OMIM 608637). Cells of these patients demonstrated impaired sulfation of chondroitin chains due to defective chondroitin 6-sulfotransferase activity . Patients with variants in CHST3 have dislocated joints, club foot, arthrogryposis multiplex congenital and in some instance hearing loss.
We report here the identification of biallelic variants in CHST3 in three different consanguineous families with multiple individuals affected by SED with joint dislocations. Affected individuals in all families had severe short statures, joint dislocations, progressive scoliosis and deformed feet.
Patients and methods
This study was conducted after obtaining approvals from the Institutional Review Board of Institute of Biomedical and Genetic Engineering, Islamabad and Institutional Review Board of School of Biological Sciences, University of the Punjab, Lahore. Written informed consents were obtained from all families who participated in this study.
Three consanguineous families (SND-65, SND-17 and NAD-05) with multiple affected individuals were included in the present study. Detailed history about disease onset and progress was obtained and documented. Heights of affected and unaffected individuals were measured. Photographs and radiographs were obtained wherever possible. Blood samples were obtained from all participating individuals and DNA was extracted using either standard salting out or phenol chloroform methods.
For families SND-65 and SND-17 (Fig. 1A-D) whole exome sequencing was performed for index patients. Nimblegen SeqCap EZ Exome v3 kit was used for targeted enrichment using genomic DNA of index patients from the three families followed by barcoding for sequencing on a single lane of a multiplexed 2 × 151 bp sequencing run on the Illumina HiSeq 2000 platform. Reads with mean coverage of 38 per target base were obtained. Mapping of reads were done using BWA v0.7.17  and variants were called using the GATK v2 Unified Genotyper. Variants with minor allele frequency ≤ 1% in 1000 Genomes, HapMap and ExAC populations were kept while filtering exome sequencing data.
For family NAD-05 (Fig. 1E), whole exome sequencing (WES) was performed on DNA sample from individual IV:3 (Fig. 1F-H) using Agilent V4 enrichment kit (Agilent Technologies, Santa Clara, CA). 50× coverage of paired-end reads was obtained on an Illumina Hi-Seq 2000 sequencer (Otogenetics, Norcross, GA). The reads were mapped to UCSC hg19 reference human genome (http://genome.ucsc.edu/) and wANNOVAR (http://wannovar.usc.edu/) was used for annotation of variants. All heterozygous variants and variants with a minor allele frequency (MAF) greater than 0.01 in public databases (dbSNP database, GnomAD, Exome Aggregation Consortium (ExAC), 1000 Genomes and 6500 exon sequence project) were excluded. Exonic and splice site variants were considered for downstream analysis.
Online prediction tools including SIFT , Polyphen2  Mutation Taster (http://www.mutationtaster.org/), Mutation Assessor (http://mutationassessor.org/r3/) Revel score and M.CAP (http://bejerano.stanford.edu/mcap/) were used for assessing pathogenicity of identified variants. Three dimensional structural changes at the protein level were predicted using online tool Have your Protein Explained (HOPE) (http://www.cmbi.ru.nl/hope/input).
The segregation of variant with phenotype in families was checked by Sanger sequencing. Primer3.0 was used for designing Primers (https://bioinfo.ut.ee/primer3–0.4.0/).
Phenotypic findings in family SND-65
Clinical assessments were performed at National Institute of Rehabilitation Medicine (NIRM) and Polyclinic Hospital Islamabad, Pakistan. Family SND-65 comprised of eight affected individuals from different branches of the family (Fig. 1A-C). Two affected individuals were deceased at the time of sampling. All affected individuals had typical features of Larsen syndrome including severe short stature (height SD score < 7.7) (Table 1), deformed and dislocated hips and knee joints, complex arthrogryposis multiplex congenita and facial dysmorphism. The disease severity was progressive with age as older patients had severe phenotype. Patient IV:3 was operated to straighten the feet and is currently wheelchair bound.
Radiological examination in individual IV:6 at 13 years of age showed mild thoracolumbar scoliosis, spondylolisthesis of the lower lumbar spine, generalized disk space narrowing with irregular endplates, relative narrowing of the interpecidular distance of the mid-lumbar spine, subluxation of the hip joints with flat proximal femoral epiphyses, prominent lesser trochanter, and severe genu valgum with dysplastic distal femoral and proximal tibial epiphyses (Fig. 1B). Echocardiogram of individual IV:6 if SND-65 indicated normal cardiac morphology. Audiometry of the same individual showed mild bilateral hearing loss although he apparently had normal hearing ability and did not complain of impairment in hearing.
Phenotypic findings in family SND-17
The clinical examination and radiological examination of SND-17 was performed in THQ hospital Peshawar. Family SND-17 included five affected individuals born to a consanguineous couple (Fig. 1C-D). The index patient was an 8-year-old boy with short stature, mild facial dysmorphism, dislocated joints, mild arthrogryposis multiplex congenita, club foot and progressive kyphoscoliosis. All his affected siblings had similar clinical manifestations.
Radiological examination of the index patient III:7 showed degenerative joint disease of both hip joints, prominent lesser trochanters, severe genu valgum with dysplastic epiphyses of both knee joints, and supernumerary carpal bones (Fig. 1D).
Family NAD-05 comprised of two affected siblings born to consanguineous parents (Fig. 1E-H). Both affected individuals had severe short stature (height SD score < 8.7), dislocated joints, arthrogryposis multiplex congenita, mild facial dysmorphism and progressive kyphoscoliosis. Individual IV:1 used walking aids while individual IV:2 could walk on her own but with difficulty.
The affected individuals in the three families had normal cognition hearing and speech. The obligate heterozygotes were of average height and had no skeletal anomaly or clinical signs of premature osteoarthritis.
Whole exome sequencing (WES) was performed on sample from one affected individual in each of the three families. Data analysis revealed a novel homozygous missense mutation c.590 T > C;p.(Leu197Pro) in CHST3 in family SND-65 while known biallelic variants c.603C > A;p.(Tyr201Ter) and c.661C > T;p.(Arg221Cys) in CHST3 were identified in families SND-17 and NAD-05, respectively. Sanger sequencing confirmed that the three variants segregated with the disease phenotype in the respective families Supplementary Fig. 1. The variants were predicted to be pathogenic by various online prediction tools including SIFT, Mutation taster, Mutation assessor and Polyphen. c.661C > T;p.(Arg221Cys) variant in CHST3 identified in NAD-05 was absent in 150 unrelated individuals from Pakistan. Moreover, there were no other potential pathogenic variants identified in the whole exome data which segregated in the families with the disease phenotype.
Larsen syndrome or spondyloepiphyseal dysplasias are a group of genetically heterogeneous osteochondrodysplasias characterized by severe short stature, severe dislocation and deformation of joints, facial dysmorphism and progressive kyphoscoliosis. Chondrodysplasia with congenital joint dislocations, CHST3 type is an autosomal recessive form of SED with a progressive disease course leading from normal length at birth to severe short stature in adulthood . In the present study we have identified three different disease-causing variants in CHST3, including one novel variant, in three different Pakistani families.
CHST3 is located on chromosome 10 and encodes carbohydrate sulfotransferase 3 (C6ST) also called chondroitin-6-sulfotransferase 3 (C6ST). C6ST catalyzes the modifying step of chondroitin sulfate (CS) synthesis by transferring sulfate to the C-6 position of the N-acetylgalactosamine of chondroitin . Chondroitin is one of the sugar components of glycosaminoglycan that is attached to core proteins in several proteoglycans and macromolecules widely distributed throughout the body. Sulfation of glycosaminoglycan creates negative charge on its surface, which is crucial for biological function of glycosaminoglycan. C6ST is localized to the Golgi complex and CHST3 is highly expressed in heart, skeletal muscles, placenta and thymus. Murine studies demonstrate that Chst3 null mice are born at normal frequency with no obvious phenotype . However, humans with null CHST3 variants have SED [2, 9]. To date 48 different types of mutations have been identified in CHST3. (www. Hgmd.cf. ac. uk accessed May, 2022).
The CHST3 missense variant c.590 T > C;p.(Leu197Pro) identified in family SND-65 was novel and may result in production of protein with little or no functional ability. The identified CHST3 variants c.603C > A;p.(Tyr201Ter) in affected individuals of family SND-17 and c.661C > T;p.(Arg221Cys) in NAD-05 patients have been previously reported in affected individuals of one Spanish and two unrelated Asian families with SED [9, 10]. Unfortunately the 3D structure model of the CHST3 protein is not available and using HOPE preliminary Protein structure modelling analysis for p.(Leu197Pro) variant predicted that the Proline197 residue in mutant protein is present in helical region of protein and will effect structure of protein severely as Proline produces kink when present in helix. Patients with variants in CHST3 were initially diagnosed with Larsen syndrome, chondrodysplasia with multiple dislocations or spondyloepiphyseal dysplasia . Our patients had similar clinical features including joint dislocation, club foot, kyphosis and dysplastic elbow joint with restricted extension as documented for the previous cases. Some spondyloepiphyseal dysplasia patients harboring CHST3 variants have also demonstrated multiple heart valve deformities [10, 11]. Contrarily, echocardiography of index patient in SND-65 reveals normal cardiac features. The codons 197 and 221 are evolutionary conserved and are part of sulfotrasnferase domain of CHST3 (Fig. 2 A-B). They might affect the catalytic activity of CHST3.
A truncating variant c.802G > T;p.(Glu268Ter) in CHST3 was previously reported in a large consanguineous family from Pakistan . Affected individuals in that family had mixed hearing loss in addition to typical features of spondyloepiphyseal dysplasia. Similarly, a variable degree of hearing loss has also been observed in some other reported SED patients with CHST3 variants but with phenotypic variability among individuals harboring the same CHST3 allele. The phenotypic variability among patients with CHST3 variant is attributed to ethnicity and geographic origin [1, 9]. In contrast to these previous reports, affected individuals in the present study did not have any signs of hearing loss except for 1 patient in SND-65. This study indicates phenotypic heterogeneity among individuals with CHST3 variants even from the same geographic background.
In conclusion, we report here three Pakistani families comprising multiple individuals with SED-CHST3 type. All subjects harbored homozygous variants in the CHST3 gene; two of the variants have been previously reported in patients in different populations while one variant was novel. Our study expands the phenotypic and genotypic spectrum of this rare skeletal dysplasia.
Availability of data and materials
Data can be obtained from corresponding author upon reasonable request. We have deposited the variant sequence in LOVD (ID:0000869250). https://databases.lovd.nl/shared/variants/0000869250#00005158.
Mortier GR, Cohn DH, Cormier-Daire V, Hall C, Krakow D, Mundlos S, et al. Nosology and classification of genetic skeletal disorders: 2019 revision. Am J Med Genet A. 2019;179(12):2393–419.
Thiele H, Sakano M, Kitagawa H, Sugahara K, Rajab A, Höhne W, et al. Loss of chondroitin 6-O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement. Proc Natl Acad Sci. 2004;101(27):10155–60.
Consortium GP. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491(7422):56–65.
Sim N-L, Kumar P, Hu J, Henikoff S, Schneider G, Ng PC. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 2012;40(W1):W452–W7.
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248–9.
Rajab A, Kunze J, Mundlos S. Spondyloepiphyseal dysplasia Omani type: a new recessive type of SED with progressive spinal involvement. Am J Med Genet A. 2004;126(4):413–9.
Tsutsumi K, Shimakawa H, Kitagawa H, Sugahara K. Functional expression and genomic structure of human chondroitin 6-sulfotransferase 1. FEBS Lett. 1998;441(2):235–41.
Uchimura K, Kadomatsu K, Nishimura H, Muramatsu H, Nakamura E, Kurosawa N, et al. Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates. J Biol Chem. 2002;277(2):1443–50.
Hermanns P, Unger S, Rossi A, Perez-Aytes A, Cortina H, Bonafé L, et al. Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and Humero-spinal Dysostosis. Am J Hum Genet. 2008;82(6):1368–74.
Unger S, Lausch E, Rossi A, Mégarbané A, Sillence D, Alcausin M, et al. Phenotypic features of carbohydrate sulfotransferase 3 (CHST3) deficiency in 24 patients: congenital dislocations and vertebral changes as principal diagnostic features. Am J Med Genet A. 2010;152(10):2543–9.
Tuysuz B, Mizumoto S, Sugahara K, Celebi A, Mundlos S, Turkmen S. Omani-type spondyloepiphyseal dysplasia with cardiac involvement caused by a missense mutation in CHST3. Clin Genet. 2009;75(4):375–83.
Waryah AM, Shahzad M, Shaikh H, Sheikh SA, Channa NA, Hufnagel RB, et al. A novel CHST3 allele associated with spondyloepiphyseal dysplasia and hearing loss in Pakistani kindred. Clin Genet. 2016;90(1):90–5.
We are thankful to the families for participating in this study. The study was partially funded by Koshish Foundation USA (SN).
This study was partially funded by Koshish Foundation (grant to Sadaf Naz).
Ethics approval and consent to participate
This study was conducted after obtaining approvals from the Institutional Review Board of Institute of Biomedical and Genetic Engineering, Islamabad and Institutional Review Board of School of Biological Sciences, University of the Punjab, Lahore. Written informed consents were obtained from the participants and parents for their minor children. All methods were performed in accordance with relevant guidelines and regulations.
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Written informed consents were obtained from patients or their parents.
The authors declare no conflict of interest.
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Chromatograms of CHST3 sequence in families SND-65, SND-17 and NAD-05. (A) Partial chromatograms of sequence of CHST3 of family SND-65. Arrows indicate point of mutation, c.590 T > C;p.(Leu197Pro). (B) Partial chromatograms of sequence of CHST3 of family SND-17. Arrows indicate point of mutation, c.603C > A;p.(Tyr201Ter). (C) Partial chromatograms of sequence of CHST3 of family NAD-05. Arrows indicate point of mutation, c.661C > T;p.(Arg221Cys).
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Kausar, M., Ain, N.U., Hayat, F. et al. Biallelic variants in CHST3 cause Spondyloepiphyseal dysplasia with joint dislocations in three Pakistani kindreds. BMC Musculoskelet Disord 23, 818 (2022). https://doi.org/10.1186/s12891-022-05719-6