Fetal structural abnormalities emerge in approximately 3.0% of pregnancies, which can be related to all types of genetic variants (Persson et al., 2017; Lord et al., 2019). Karyotyping and chromosomal microarray analysis are recommended as the preferred diagnostic methods for fetal structural abnormalities (International Society for Prenatal Diagnosis, 2018). However, more than 60% of fetal structural abnormalities cannot be explained by chromosomal karyotyping and microarray analysis (Wapner et al., 2012). Recently, whole-exome sequencing (WES) has been confirmed to be a valuable diagnostic approach for explicating the underlying genetic etiology for many likely Mendelian disorders (Petrovski et al., 2019).

The Ehlers–Danlos syndromes (EDS) are a group of heritable connective tissue diseases involving at least 17 genes and 13 subtypes, with various symptoms, characteristically joint hypermobility, skin hyperextensibility, and tissue fragility (Malfait et al., 2017). Musculocontractural Ehlers–Danlos syndrome (mcEDS) is a subtype of EDS caused by homozygous or compound heterozygous mutations of the carbohydrate sulfotransferase 14 (CHST14) or dermatan sulfate epimerase gene (Malfait et al., 2010). Three major clinical criteria are defined for the diagnosis of mcEDS, including 1) congenital multiple contractures, typically adduction-flexion contractures, and/or talipes equinovarus (clubfoot); 2) characteristic craniofacial features; and 3) characteristic cutaneous features, for example, skin hyperextensibility, easy bruisability, and skin fragility (Malfait et al., 2017).

The diagnosis of EDS once mainly relied on clinical features (Beighton et al., 1988). Then, in 1997, the biochemical and molecular bases were required to classify EDS (Beighton et al., 1998). A molecular confirmation is very important for the diagnosis and counseling in view of the overlap of clinical symptoms between different EDS subtypes. Recently, it has been recommended that molecular detection should base on next-generation sequencing technologies, such as copy number variation (CNV) detection, WES, and whole-genome sequencing (WGS) (Malfait et al., 2017). Prenatal diagnosis of mcEDS is more difficult than postnatal diagnosis, as the craniofacial and cutaneous features have not been represented completely. Up till now, prenatal mcEDS has not yet been reported. We herein introduce an mcEDS case diagnosed by prenatal WES and Sanger sequencing.

Next-generation sequencing such as CNV-seq, WES, and WGS has been widely used in clinical practice in the last decade. It has been reported that WES-trio achieved a diagnostic rate of 40% in diagnosing genetic disorders, which was almost as high as that of WGS-trio (42%) (Lord et al., 2019). Seven genes implicating stillbirth were identified by WES, with a detection rate of 6.1% (Stanley et al., 2020). Therefore, WES has recently been recommended to be used in prenatal clinical practice to uncover the underlying genetic causes of fetal structural anomalies while abnormal karyotype and pathogenic CNV had been excluded (Petrovski et al., 2019).

CHST14 gene is located in number 15 chromosome (15q15.1), involving only one exon and encoding N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which plays an essential role in the biosynthesis of proteoglycans (CHST14 carbohydrate sulfotransferase 14 [Homo sapiens (human)] - Gene - NCBI (nih.gov)). Proteoglycans are abundant in the extracellular matrix and important in a wide range of physiological functions, such as interacting with collagen (Malfait et al., 2020). Pathogenetic mutations in the CHST14 gene result in deficiency of D4ST1, which consequently leads to the decrease of proteoglycans and further abnormal regulation of collagen fibrils assembly and finally gives rise to the mcEDS (Dundar et al., 2009; Malfait et al., 2020).

To our best knowledge, at least 26 variants of the CHST14 gene have been reported. However, no apparent relationship between genotype and phenotype is noted (Minatogawa et al., 2021). Dundar et al. (2009) discovered a 1 bp deletion (c.145_146 delG), a missense mutation (c.638G>C), and a complex allele (c.404C>G; 410T>A) in CHST14 in Australian Turks with thumb-clubfoot Syndrome. Miyake et al. (2010) reported four mutations (c.842C>T p.P281L, c.866G>C p.C289S, c.878A>G p.Y293C, c.205A>T p.K69*) of the CHST14 gene in six Japanese patients with Kosho type EDS. In fact, thumb-clubfoot syndrome and Kosho type EDS have a common clinical condition, so they are termed mcEDS (Malfait et al., 2010).

In this study, novel and reported mutations in CHST14 were detected in a fetus with adduction flexion in the feet and renal pelvis in the kidneys. A likely pathogenic mutation [NM_130468.3 c.958C>T (p.Arg320*)], which was also found in the mother, had been reported previously by Minatogawa et al. (2021) in a study involving 66 mcEDS patients. A novel variation [NM_130468.3 c.896A>G (p.Tyr299Cys)], which was also found in the father, was evaluated as an uncertain significance mutation according to the ACMG guidelines. However, it was predicted to be a disease-causing or probably damaging mutation in in silico analyses. The two detected variants are located in the middle of the sulfotransferase domain and presumably result in partial or complete loss of function of D4ST1 (Minatogawa et al., 2021).

The couple decided to terminate this pregnancy after genetic counseling. Clubfeet (Supplementary Figure S3) were confirmed by autopsy, and this was consistent with the ultrasound results. The autopsy record about kidneys was unknown. Clubfoot was one of the three major criteria for diagnosing mcEDS and 95% (59/62) mcEDS patients developed clubfoot, while renal structural abnormalities were not observed (Minatogawa et al., 2021). By considering the clinical symptom (clubfeet) and the molecular detective results, an alternative diagnosis of mcEDS was made to the fetus. It was clear that the fetus inherited the two mutations from both parents and became a carrier of compound heterozygous mutations of the CHST14 gene. It appears highly likely that the structural abnormalities, especially clubfeet, are caused by the two mutations. However, further functional studies, such as cell experiments, are needed to support the assumption.

In conclusion, we identified a novel mutation [NM_130468.3 c.896A>G (p.Tyr299Cys)] and a reported likely pathogenic mutation [NM_130468.3 c.958C>T (p.Arg320*)] in the CHST14 gene of by WES prenatally, which can perhaps be claimed as the potential genetic etiology of the fetal structural abnormalities.