Homozygous splice site variant affecting the first von Willebrand factor A domain of COL12A1 in a patient with myopathic Ehlers‐Danlos syndrome

Myopathic Ehlers‐Danlos syndrome (mEDS) is a subtype of EDS that is caused by abnormalities in COL12A1. Up‐to‐date, 24 patients from 15 families with mEDS have been reported, with 14 families showing inheritance in an autosomal dominant manner and one family in an autosomal recessive manner. We encountered an additional patient with autosomal recessive mEDS. The patient is a 47‐year‐old Japanese man, born to consanguineous parents with no related features of mEDS. After birth, he presented with hypotonia, weak spontaneous movements, scoliosis, and torticollis. He had soft palms but no skin hyperextensibility or fragility. Progressive scoliosis, undescended testes, and muscular torticollis required surgery. During adulthood, he worked normally and had no physical concerns. Clinical exome analysis revealed a novel homozygous variant in COL12A1 (NM_004370.6:c.395‐1G > A) at the splice acceptor site of exon 6, leading to in‐frame skipping of exon 6. The patient was diagnosed with mEDS. The milder manifestations in the current patient compared with previously reported patients with mEDS might be related to the site of the variant. The variant is located in the genomic region encoding the first von Willebrand factor A domain, which affects only the long isoform of collagen XII, in contrast to the variants in previously reported mEDS patients that affected both the long and short isoforms. Further studies are needed to delineate comprehensive genotype–phenotype correlation of the disorder.


| INTRODUCTION
The Ehlers-Danlos syndromes (EDS) are a heterogenous group of heritable connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility.In 2017, the new International Classification on EDS and the relevant diagnostic criteria were proposed on the basis of a comprehensive review of EDS-related literature including studies on novel subtypes (Brady et al., 2017;Malfait et al., 2017).A subtype caused by heterozygous or biallelic variants in COL12A1, which encodes procollagen XII, was included and designated as "myopathic EDS (mEDS)" (Brady et al., 2017;Malfait et al., 2017).Major criteria include congenital muscle hypotonia and/or muscle atrophy that improves with age, proximal joint contractures (knee, hip, and elbow), and hypermobility of distal joints.Minor criteria include soft, doughy skin, atrophic scarring, motor developmental delay, and myopathy on muscle biopsy (Malfait et al., 2017).At present, 24 published patients from 15 families with mEDS have been reported, with 14 families showing inheritance in an autosomal dominant manner and one family in an autosomal recessive manner (Table 1) (Zou et al., 2014;Hicks et al., 2014;Punetha et al., 2017;Witting et al., 2018;Mohassel et al., 2019;Delbaere et al., 2020;Araújo & Antunes, 2021;Coppens et al., 2022).
Collagen XII is a homotrimer, containing three α1(XII)-chains that are encoded by COL12A1.It consists of two short triple helical domains (COL1 and COL2) separated by two noncollagenous domains (NC1 and NC2) as well as large amino-terminal noncollagenous domains (NC3).The NC3 domain comprises one thrombospondin N-terminal domain (TSPN), four von Willebrand factor A domains (vWA), and 18 fibronectin type III repeats (Delbaere et al., 2020), and it has splice variants resulting in long and short isoforms.The long isoforms included all motifs described above, whereas the short isoform lacks first two vWA and 8 fibronectin type III repeats (Böhme et al., 1995).Collagen XII molecules assemble as homotrimers as well as short and long isoform heterotrimers (Chiquet et al., 2014;Koch et al., 1995).The long isoform is predominant at early development of the embryo in mice (ED7 and 11) and the short isoform is predominant at later developmental stages (ED15 and 17); after birth, the long transcript continues to be expressed in only a few dense connective tissue (bone, tendon, ligament, dermis, cornea, blood vessel wall, and meninges) (Böhme et al., 1995).
Here, we report the second patient from another family with a recessively-inherited type of mEDS who was found to have a novel biallelic variant in COL12A1.
Total RNA was extracted from B-lymphoblastoid cell lines using a QIAamp RNA Blood Mini Kit, and was treated with an RNase-Free DNase Set (Qiagen).Total RNA was reverse-transcribed using a Pri-meScript RT reagent Kit (TaKaRa Bio, Shiga, Japan).Gene-specific primers were designed as follows: the forward primer at exon 4 of COL12A1, 5 0 -ACCCTTTCAGCTAGTACCACTG-3 0 , and the reverse primer at exon 7 of COL12A1, 5 0 -GAAAGCCAACTCTTGCCCCA-3 0 .Sanger sequencing was performed on an ABI 3130xl genetic analyzer using a BigDye Direct Cycle Sequencing Kit and a BigDye XTerminator Purification Kit (Thermo Fisher Scientific), according to the manufacturer's instructions.

| Case description
The current patient, a 47-year-old Japanese man, was the second child of consanguineous parents.His mother suddenly died at the age of 69 years of an unknown cause.His father was 76 years old and developed an aortic dissection.His older brother was healthy.The patient was delivered by spontaneous vaginal delivery.Right after birth, he was noted to have hypotonia, scoliosis, weak spontaneous movements, and a torticollis.He was suspected to have Marfan syndrome at the age of 1 year and had regular cardiovascular check-ups thereafter.Because of hypotonia, he showed motor developmental delay and was able to walk unassisted at the age of 3 years.His physical features included a slender build and a triangular face with short palpebral fissures, a small nose, a small mouth, and large ears (Figure 1a).He underwent surgery for undescended testes at preschool age.He often experienced bilateral knee dislocations.He underwent surgery for congenital muscular torticollis at the age of 11 years.He showed kyphosis, which developed into progressive scoliosis, requiring fixation surgery when he was a junior high school student.He was not good at running and exercise in his childhood, but his cognitive development was normal.When he was a lower grade elementary school student, he belonged to the special class for physically handicapped children.
He entered a regular class thereafter with no apparent difficulty in physical activities.He had a short stature, and no cardiovascular abnormalities were noted in his childhood to adolescence.
When the patient was referred to us at the age of 47 years for genetic confirmation of the diagnosis, his weight was 50.5 kg (À1.9 SD), height was 158.4 cm (À2.2 SD), occipitofrontal circumference was 58.0 cm (+0.3 SD), and arm span was 160.0 cm.No visual impairment was noted.His craniofacial features were less characteristic than those in his childhood (Figure 1b).He had a high palate, hypermobile small joints (Figure 1c), slender fingers (Figure 1d, e), pes planus, and T A B L E 1 Clinical and molecular features of previously reported patients with mEDS.Note: Blank, data not available.Abbreviations: AD, autosomal dominant; AR, autosomal recessive; ASD, atrial septal defect; B, at birth; Ch, childhood; F, female; M, male; Nb, newborn; UL, upper limb; VSD, ventricular septal defect.
hallux valgus (Figure 1f).He had a slender build (Figure 1g).His skin was not hyperextensible, fragile, or bruisable.Radiological examinations showed a deformed cervical spine (Figure 1h), a brachycephaly (Figure 1i), bilateral long deformed 5th fingers (Figure 1j, k), narrow feet with hallux valgus (Figure 1l), severe scoliosis persisting after surgical fixation (Figure 1m), and an asymmetric pelvis (Figure 1n).was confirmed by standard Sanger sequencing (Figure 2a).The variant was classified as PVS1_Strong according to ClinGen SVI recommendation (Abou Tayoun et al., 2018), based on the criteria as follows: (1) the one at a GT-AG 1,2 splice site, (2) the one exerting exon skipping or using of a cryptic splice site that preserves reading frame, and (3) the one at a truncated/altered region that is critical to protein according 2015 ACMG/AMP guidelines.Therefore, the variant was classified as pathogenic (PS3, PVS1_Strong, PM2_Supporting, and PM3_Supporting), in accordance with the 2015 ACMG/AMP guidelines and ClinGen SVI recommendations.

| DISCUSSION
The patient described in this report was considered to have mEDS caused by a homozygous splice site variant leading to skipping of exon 6.His main physical issues occurred in his childhood, including hypotonia with delayed motor development as well as progressive scoliosis, undescended testes, and congenital muscular torticollis, which required surgery.
Table 1 summarizes information on the previously reported patients, including the current patient, who were clinically consistent with mEDS and were found to have relevant abnormalities in COL12A1.Zou et al. (2014) reported three patients from two families who were found to have abnormalities in COL12A1.Subsequent reports described a total of 22 patients from 14 families who were considered to have mEDS based on the presence of abnormalities in COL12A1 and clinical features consistent with mEDS (Hicks et al., 2014;Punetha et al., 2017;Witting et al., 2018;Delbaere et al., 2020;Araújo & Antunes, 2021;Coppens et al., 2022), in accordance with the 2017 International Classification on the EDS (Malfait et al., 2017).One family (three patients) described by Hicks ac.jp/202008/downloads#variant) (Tadaka et al., 2019).(Zou et al., 2014).All variants in the autosomal dominant-type mEDS families were localized in the region encoding the long isoform and the short isoform (Hicks et al., 2014;Zou et al., 2014;Punetha et al., 2017;Witting et al., 2018;Delbaere et al., 2020;Araújo & Antunes, 2021;Coppens et al., 2022).These A long transcript, predominantly expressed in the early developmental stage, is restricted after birth to dense connective tissues such as bone, tendon, ligament, dermis, cornea, blood vessel wall, and meninges (Böhme et al., 1995).Mild muscular manifestations (hypotonia only in his childhood) and progressive spinal deformity (scoliosis requiring surgery) in the current patient might be related to the character of the variant (homozygous partial defect of the first vWA) affecting only the long isoform, which suggests the importance of the long isoform in the development of musculoskeletal system as well as the short isoform.
In conclusion, the current patient is the second case with autosomal recessive mEDS.His milder manifestations compared with reported patients might be related to the character of the variant, which affects only the long isoform of collagen XII, in contrast to previously reported patients with mEDS (variants in autosomal dominant patients affecting the long and short isoforms and a null variant in autosomal recessive patients).Further studies are needed to delineate the comprehensive genotype-phenotype correlation of the disorder.
Cardiac ultrasonography detected no aortic root dilatation or mitral valve prolapse/regurgitation.Manual muscle testing showed normal proximal or distal muscle strength.Respiratory function was not assessed because the patient feels no difficulties in daily activities or normal physical activities.Serum creatinine kinase (CK) value was normal (93 U/L).He worked at a company with no physical concerns.In view of these findings, he was suspected to have a subtype of EDS rather than Marfan syndrome.3.2 | Variant detection and mRNA analyses No pathogenic variant was detected through panel-based nextgeneration sequencing for the 17 genes.Clinical exome sequencing using the TruSight One Sequencing Panel revealed a novel homozygous variant (NM_004370.6:c.395-1G> A) in COL12A1; the variant F I G U R E 1 Clinical photographs of the patient in his early childhood (a) and at the age of 47 years (b-g).Whole body radiographs of the patient at the age of 47 years (h-n).
function; and was classified as PM3_Supporting (homozygous occurrence) according to ClinGen SVI recommendation for PM3 version 1.0.Copy number of COL12A1 was found to be normal (data not shown).The variant was not registered in gnomAD, which was classified as PM2_Supporting according to ClinGen SVI recommendation for PM2 version 1.0.RT-PCR and Sanger sequencing of COL12A1 cDNA from lymphoblastoid cell lines in the patient identified an abnormal COL12A1 transcript with skipping of exon 6 (Figure 2b, c).It was expected to lead to partial defect of the first vWA in collagen XII (p.Lys132_Gly220delinsArg) (Figure 2d), which was judged as PS3 F I G U R E 2 (a) Visualization of next-generation sequencing data with Integrative Genomics Viewer (top) and Sanger sequencing electropherogram of the variant (bottom).The minus-strand is shown.(b) Gel electrophoresis of RT-PCR products (upper) and schematic representation of the aberrantly spliced mRNA resulting from the splice site variant (lower).Gene-specific primers were shown on the schematic as red arrows.M: GeneRuler 100 bp DNA Ladder Plus (Fermentas); P: patient; C: healthy control.The variation (c.395-1G > A) at the splice acceptor site of exon 6, leading to skipping of exon 6, is shown in red font.(c) Sanger sequencing of COL12A1 cDNA.(d) The distribution of COL12A1 variants in 16 families with mEDS shown on the schematic representation of the exons of the long mRNA transcript (upper) and the protein domains of the collagen XII long isoform (lower).Exons involved in the long mRNA transcript (NM_004370.6) and not in the short mRNA transcript (NM_080645.3) are highlighted as gray boxes."1" and "9092" indicate the first and the last nucleotide positions of the coding region of the mRNA, respectively.hom: homozygote; f1-f16: families 1-16.(e) A schematic diagram of the collagen XII structure (long isoform and short isoform) and the sites of the variants found in the current patient as well as in previously reported patients.
et al. (2014) was excluded because the frequency of the detected variant (c.5893C > T,p.Arg1965Cys) was high (1/4009 as shown by gno-mAD).One patient described by Delbaere et al. (2020) was excluded because the frequency of the detected variant (c.5587C > T,p.Arg1863Cys) was high (1/2095) according to ToMMo 8.3KJPN Genotype Frequency Panel (v20200831) (https://jmorp.megabank.tohoku. variants, affecting both the long isoform and the short isoform, might cause dysfunction of collagen XII through disrupting its collagen structure.In contrast, the variant detected in the current patient was a novel homozygous splice site variant (NM_004370.6:c.395-1G> A) leading to exon 6 skipping.It was expected to lead to partial defect of the first vWA domain in collagen XII (p.Lys132_Gly220delinsArg); this variant would only affect the long isoform (Figure2d, e).