Skip to main content
NCBI home page
Molecular Genetics & Genomic Medicine logoLink to Molecular Genetics & Genomic Medicine
. 2023 Dec 11;12(1):e2331. doi: 10.1002/mgg3.2331

Exome sequencing‐aided precise diagnosis of four families with type I Stickler syndrome

Runyi Tian 1,2, Ping Tong 3, Yuhong He 2, Liyu Zang 1,4,5, Shimin Zhou 1,4,5, Qi Tian 1,4,5,✉
PMCID: PMC10767595  PMID: 38073514

Abstract

Background

Stickler syndrome is a multisystemic disorder characterized by ophthalmological and non‐ophthalmological abnormalities, frequently misdiagnosed due to high clinical heterogeneity. Stickler syndrome type I (STL1) is predominantly caused by mutations in the COL2A1 gene.

Methods

Exome sequencing and co‐segregation analysis were utilized to scrutinize 35 families with high myopia, and pathogenic mutations were identified. Mutant COL2A1 was overexpressed in cells for mechanistic study. A retrospective genotype–phenotype correlation analysis was further conducted.

Results

Two novel pathogenic mutations (c.2895+1G>C and c.3505G>A (p.Val1169Ile)) and two reported mutations (c.1597C>T (p.Arg533*) and c.1693C>T (p.Arg565Cys)) in COL2A1 were identified causing STL1. These mutations are all in the G‐X‐Y triplet, and c.2895+1G>C contributed to aberrant RNA splicing. COL2A1 mutants tended to form large aggregates in the endoplasmic reticulum (ER) and elevated ER stress. Additionally, mutations c.550G>A (p.Ala184Thr) and c.2806G>A (p.Gly936Ser) in COL2A1 were found in high myopia families, but were likely benign, although c.2806G>A (p.Gly936Ser) is on G‐X‐Y triplet. Moreover, genotype–phenotype correlation analysis revealed that mutations in exon 2 mainly contribute to retinal detachment, whereas mutations in the collagen alpha‐1 chain region of COL2A1 tend to cause non‐ophthalmologic symptoms.

Conclusion

This study broadens the COL2A1 gene mutation spectrum, provides evidence for ER stress caused by pathogenic COL2A1 mutations and highlights the importance of non‐ophthalmological examination in clinical diagnosis of high myopia.

Keywords: COL2A1, ER stress, genotype–phenotype correlation, novel mutations, Stickler syndrome

Our study identified two novel STL1‐related COL2A1 mutations causing ER stress, and highlights the importance of non‐ophthalmological examination in clinical diagnosis of high myopia.

graphic file with name MGG3-12-e2331-g005.jpg

1. INTRODUCTION

Stickler syndrome was named by Gunnar B. Stickler et al., who first described this systemic connective tissue disorder (Stickler et al., 1965). Stickler syndrome is characterized by ophthalmologic, orofacial, auditory, and musculoskeletal manifestations, including high myopia, congenital cataracts, glaucoma, vitreous abnormalities, retinal detachment, midfacial hypoplasia, hearing loss, hypermobile joints, arthritis, and spinal deformation (Snead et al., 2011; Stickler et al., 2001). The prevalence of Stickler syndrome among neonates is approximately 1/7500–1/9000 (Robin et al., 1993). Based on the systemic abnormalities involved and the causal collagen genes, Stickler syndrome is classified into the following five types: STL1 (MIM no. 108300, caused by COL2A1 gene mutations); STL2 (MIM no. 604841, caused by COL11A1 gene mutations); STL3 (MIM no. 184840, caused by COL11A2 gene mutations); STL4 (MIM no. 614134, caused by COL9A1 gene mutations); and STL5 (MIM no. 614284, caused by COL9A2 gene mutations).

STL1 is the predominant type compared with the others, accounting for approximately 80%–90% of cases) (Hoornaert et al., 2010; Snead & Yates, 1999). STL1 is a classic autosomal dominant‐inherited disorder. COL2A1, which encodes the alpha‐1 chain of type II collagen, is mainly expressed in collagen‐rich organs and tissues, including the vitreous, inner ear, and cartilage (Rukavina et al., 2014). Three pro‐alpha‐1 chains encoded by COL2A1 tangle to form procollagen molecules, which are then processed and crosslinked to form mature type II collagen fibrils (Robins, 2006). Mature type II collagen fibrils are enriched with long uninterrupted G‐X‐Y triplet repeats. The first position of the triplet is glycine, while the X and Y positions include but are not limited to proline and hydroxyproline (Brodsky & Persikov, 2005). The triple helix folding domain, characterized by the G‐X‐Y triplet located in the collagen alpha‐1 chain, plays a pivotal role in helix folding and stability (Engel & Bächinger, 2005). COL2A1 has two isoforms. One is long type IIA, and the other is short type IIB which is due to the alternative splicing of exon 2. Type IIA is predominantly expressed in the eye, and IIB is ubiquitously expressed (Reardon et al., 2000; Sandell et al., 1994). The expression pattern of IIA and IIB gives rise to ophthalmologic, auditory, and musculoskeletal system abnormalities caused by COL2A1 gene mutations with high clinical heterogeneity (Donoso et al., 2003; McAlinden et al., 2008; Richards et al., 2010; Robin et al., 1993).

Ophthalmologic change is a common feature of STL1. Patients tend to seek treatment for visual problems such as high myopia, cataracts, and retinal detachment. However, misdiagnosis of STL1 as high myopia leads to improper treatment, consequently impeding effective intervention and therapy. A combination of clinical and genetic examinations is a promising strategy for distinguishing STL1 from non‐syndromic high myopia, further benefiting pre‐treatment, genetic counseling, and eugenics.

In this study, we utilized exome sequencing to screen probands from 35 families with high myopia and identified four STL1‐causing variants, including two novel pathogenic variants in COL2A1. The COL2A1 protein carrying STL1‐associated missense mutations tended to form aggregates and induced ER stress and elevated unfolded protein response (UPR). Intriguingly, the c.2806G>A mutation located in the G‐X‐Y triplet, which is crucial for helix formation, was likely benign. Moreover, a retrospective analysis of genotype–phenotype correlation indicated that mutations in exon 2 mainly caused retinal detachment, whereas mutations in the collagen alpha‐1 chain region of the COL2A1 protein contributed more to non‐ophthalmologic symptoms. Our study expands the knowledge of the COL2A1 mutation spectrum and provides evidence that not all variations in the G‐X‐Y triplet are detrimental. This study highlights the importance of combining clinical and genetic evaluations in patients with high myopia phenotypes for precise diagnosis and treatment.

2. MATERIALS AND METHODS

2.1. Ethical compliance

This study was approved by the Institutional Review Board (IRB) of the School of Life Sciences, Central South University, Changsha, China. All procedures followed the relevant policies in China and adhered to the Declaration of Helsinki. All subjects who participated in this study thoroughly read and signed an informed consent form.

2.2. Subjects

In this study, the adult subjects with refractive error (RE) ≤ −5.00D were defined as high myopia patients using the World Health Organization (WHO) recommended threshold (Flitcroft et al., 2019). The threshold for subjects younger than 15 years was set to RE ≤ −3.00D. We recruited a total of 341 subjects from 35 families with large congenital high myopia from different regions in China. The 341 subjects included 171 patients with high myopia and 170 asymptomatic individuals. Pedigree analysis showed a dominant inheritance pattern in 34 families and a recessive pattern in one. 200 sporadic subjects (79 males and 121 females) with high myopia were recruited according to the criteria above. After obtaining informed consent, blood samples were collected for analysis from all subjects.

2.3. Clinical evaluations

The probands of families with high‐grade myopia underwent complete ophthalmologic examinations of visual acuity by LogMAR chart, refraction by auto‐refractor, lens and vitreous by slit lamp, intraocular pressure by tonometry, retina by ophthalmoscope, and axial length by an A‐scan ultrasound device. The examinations were conducted at the 2nd Xiangya Hospital of Central South University in China by an experienced ophthalmologist. After genetic analysis, additional symptom examinations were performed on patients with COL2A1 mutations in the facial, auditory, and musculoskeletal systems, including flattened face, cleft palate, hearing loss, hypermobile joints, and arthritis.

2.4. Genetic analyses

Genomic DNA (gDNA) was extracted from the venous blood of all subjects. Next, exome sequencing was performed on the gDNA of the probands from these families. The sequencing procedure and analysis have been previously described (Tian et al., 2017). In brief, 3 μg of gDNA was sheared, and adaptors were added. The exome was captured using an Agilent SureSelectXT HumanAllExon V4+UTRs probe (Agilent, Beijing, China) and clustered by cBot, followed by sequencing on an Illumina HiSeq 2000 DNA analyzer with PE100 (Illumina, San Diego, CA, USA). The sequencing data were aligned to the GRCh37/hg19 genome reference sequence, and the variants were called using GATK and annotated using ANNOVAR software.

After screening out variants with an allele frequency >0.01 in the ExAC_asn, GnomAD_asn, and maf1000g_asn databases, all non‐synonymous variants, together with SNVs and Indels, in the consensus coding sequence (CCDs) and canonical splicing sites were reserved for further analysis.

For families with probands carrying mutations in the COL2A1 gene (isoform NM_001844.4, GenBank: L10347.1), gDNA samples from other family members were selected for Sanger sequencing to validate the mutation and perform the co‐segregation analysis in corresponding families.

2.5. Cell lines and antibodies

HeLa cells were obtained from American Type Culture Collection (Manassas, VA, USA). HeLa cells were cultured in DMEM (SH30022, HyClone, Logan, UT, USA) containing 2 mM l‐glutamine (25030, Life Technologies, Carlsbad, CA, USA) and 1 mM sodium pyruvate (11360, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (SH30084.03, HyClone, Logan, UT, USA).

Antibodies against LC3B (2775 s, rabbit), PERK (3192 s, rabbit), Phosphor‐PERK (3179 s, rabbit), calnexin (2679P), cleaved Caspase 3 (9661), and flag‐tag (14793, rabbit) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against β‐actin (A5441, mouse) was purchased from Sigma‐Aldrich (St. Louis, MI, USA), and the antibody for ATF4 (10835‐1‐AP, rabbit) was obtained from Thermo Fisher Scientific.

2.6. Minigene construction and mutation

COL2A1_exon 1–3 or exon 40–44 were assembled in two steps by overlapping extension PCR and classical restriction digestion/ligation cloning into pcDNA3.1‐his/myc‐b (V80020, Thermo Fisher Scientific, Waltham, MA, USA). All the inserts were amplified with Phusion High Fidelity polymerase (Thermo Fisher Scientific) and were inserted between the Notl and HindIII restriction sites. All the generated constructs were verified by sequencing (BioSune, Minhang, Shanghai, China).

2.7. Immunoblotting

Cells were washed with PBS and lysed with SDS buffer (2% SDS, 10% glycerol, 62.5 mM Tris HCl, pH 6.8). The protease inhibitor cocktail (Roche, Basel, Switzerland) was freshly added, and the lysates were subjected to immunoblotting with the indicated antibodies. The band intensity was quantified using Fiji software (NIH, Bethesda, MD, USA).

2.8. Immunofluorescence

Cultured cells were washed with PBS, fixed with 4% PFA for 10 min, and permeabilized with 0.1% PBST (Triton X‐100). After blocking with 5% BSA in PBS for 1 h, the cells were incubated with primary antibodies diluted in blocking solution, washed with PBS, incubated with secondary antibodies, and mounted using Fluoromount G (Southern Biotech, Birmingham, AL, USA). Images were captured using a Zeiss LSM880 confocal system (Zeiss, Jena, Germany) with a Plan‐Apochromat 63× NA 1.4 oil differential interference contrast objective lens.

2.9. Genotype–phenotype correlation analyses

To maximize the sensitivity and accuracy of the search, the terms ‘Stickler syndrome’ and ‘COL2A1’ were used in the PubMed database, and the species were filtered by ‘Humans’. The outcome papers were individually scrutinized, and those with definite phenotypes and variants were collected for the genotype–phenotype correlation analyses.

One hundred and ten articles ranging from 1989 to 2021 with detailed descriptions of patient phenotypes were analyzed. Families identified in this study were also included, and a total of 172 COL2A1 mutations in 830 patients were analyzed. Mutations and associated phenotypes were further classified and depicted.

2.10. Statistics and graphing

Differences in RE between the left and right eyes of males and females were compared using a paired t‐test with a two‐tailed p‐value. The frequency differences of the symptoms between each mutation category and the overall variations, and the mutation frequency differences between the collagen alpha‐1 chain and non‐alpha‐1 chain regions, were compared using the Chi‐square test and Fisher's exact test with a two‐tailed p‐value. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. All data were analyzed using GraphPad Prism (Version 5.01) and Microsoft Excel (Microsoft Office 2016); graphing was achieved using GraphPad Prism, Microsoft Excel, and Adobe Illustrator CS6.

3. RESULTS

3.1. Clinical and genetic studies reveal Stickler‐associated mutations in COL2A1

Thirty‐five Chinese families with high myopia were recruited. Twenty probands were females, and 15 were males, ranging from 4 to 82 years of age. The binocular average RE went from −5.75D to −29.00D, and the axial length from 24.07 to 34.45 mm. The average RE was −14.72 ± 5.69D for the left eye and −14.94 ± 6.50D for the right, with no significant difference (p = 0.7027). The RE for females and males was −14.57 ± 5.26D and −15.03 ± 6.39D, respectively, showing no statistical significance (p = 0.8218) (Table S1). After scrutinizing the exome sequencing results of probands, we identified four rare pathogenic mutations in the COL2A1 gene, c.1597C>T (p.Arg533*) in family SS1, c.1693C>T (p.Arg565Cys) in family SS2, c.3505G>A (p.Val1169Ile) in family SS3, and c.2895+1G>C in family SS4, using co‐segregation analysis (Figures 1a–c, 2a and Table S2).

FIGURE 1.

FIGURE 1

Open in a new tab

Identification of two known STL1‐causing variants on COL2A1 gene in SS1 and SS2 family. (a, b) Pedigrees for SS1 (A), SS2 (B). “□” and “○” symbols present normal male and female subjects, “■” and “●” characters stand for male and female patients, the DNA selected for exome sequencing was marked with “*” on the up‐right of the symbol, the arrow in the pedigree indicates the probands. “+” stand for wild‐type (WT) allele, and “−” refers to mutated allele. (c) Sanger sequencing results for mutations on COL2A1 in SS1 and SS2 families. The arrows in the sequence indicate the mutation site. SS1‐IV: 14 carries c.1597C>T (p.Arg533*). SS2‐IV: 2 carries c.1693C>T (p.Arg565Cys). (d) The vitreoretinal degeneration and cataract phenotypes in SS1 and SS2 families. OD, Oculus Dexter or right eye. OS, Oculus Sinister or left eye. SS1‐IV:12, SS1‐IV:14, and SS2‐III:5 showed vitreoretinal degeneration.

FIGURE 2.

FIGURE 2

Open in a new tab

Identification of two novel pathogenic variants on COL2A1 gene causing STL1 in SS3 and SS4 family. (a) Pedigrees for SS3 family and sanger sequencing results for mutation. “□” and “○” symbols present normal male and female subjects, “■” and “●” characters stand for male and female patients, the DNA selected for exome sequencing was marked with “*” on the up‐right of the symbol, the arrow in the pedigree indicates the probands. “+” stand for wild‐type allele, and “−” refers to mutated allele. The arrow in the sequence indicates the mutation site. SS3‐III: 6 carries c.3505G>A (p.Val1169Ile). (b) Pedigrees for SS4 family and sanger sequencing results for mutation. SS4‐III:2 carries a mutation at the first site on intron 42 (c.2895+1G>C). (c) Schematic representation of Minigene COL2A1 Exon 40–44 with (c.2895+1G>C) mutation. Specific amplification primers are shown as arrows. The expected COL2A1 exon 40–44 (E40–44) transcript is 398 nt. (d) Amplified RT‐PCR product. The transcript of wild‐type COL2A1 E40–44 is 398 nt, while that of (c.2895+1G>C) mutant is larger than WT. (e) Sanger sequencing of the PCR products in (d) showed that (c.2895+1G>C) mutant caused aberrant splicing, resulting in a 77 bp insertion after exon 42. This mutation disrupts the consecutive G‐X‐Y units. Mut, (c.2895+1G>C) mutant.

In family SS1, proband IV:14 was a 33‐year‐old male patient who had been seeking treatment for progressive high myopia since the age of 10 (Figure 1a). The intraocular pressure (IOP) of IV:14 was normal. Slit‐lamp examination showed a clear lens of IV:14 but with vitreoretinal degeneration (Figure 1d). A nonsense variation c.1597C>T (p.Arg533*) in the COL2A1 gene was identified by exome sequencing of proband IV:14. R533 was located on the collagen alpha‐1 chain of the COL2A1 protein and was in the Y‐position of the G‐X‐Y triplet (Figures 1c and 3c). Further, c.1597C>T (p.Arg533*) leads to a premature stop codon that may produce truncated COL2A1 that is largely devoid of the triple‐helical region. This mutation has been reported previously and was predicted to be deleterious (Liberfarb et al., 2003; Wilkin et al., 2000). The amino acids involved were highly conserved among vertebrates (Figure 3d and Table S2). In this family, six subjects had high myopia (Table S3). The left eye of patient IV:12 was blind owing to high myopia and cataracts. Co‐segregation analysis showed that six patients with high myopia and six asymptomatic persons carried the variation c.1597C>T (p.Arg533*) in this family (Figure 1a and Table S3). The penetrance of high myopia caused by this mutation was incomplete (Table S3). Facial, auditory, and musculoskeletal examinations were then performed for III:6, IV:12, and IV:14, where III:6 showed midfacial hypoplasia, and both III:6 and IV:14 had vitreoretinal degeneration and sensorineural hearing loss (Tables S3 and S4).

FIGURE 3.

FIGURE 3

Open in a new tab

The pedigree of two high myopia families and the location of six variations on COL2A1. (a, b) The pedigree and Sanger sequencing results of family HM1 (A) and HM2 (B) with high myopia. “□” and “○” symbols present normal male and female subjects, “■” and “●” characters stand for male and female patients, the DNA selected for exome sequencing was marked with “*” on the up‐right of the symbol, the arrow in the pedigree indicates the probands. “+” stand for wild‐type allele, and “−” refers to mutated allele. The arrow in the sequence indicates the mutation site. (c) The location of six variations on COL2A1 identified in this study. All mutations gather on the collagen alpha‐1 chain region. SP, signal peptide; NP, N‐terminal pro‐peptide; NT, N‐telopeptide; CT, C‐terminal pro‐peptide; CP, C‐telopeptide. GER, GRP, GPP, and GPV represent the G‐X‐Y units involved. Arrows indicate the location of mutations. Mutations locate on G‐X‐Y units are in bold. (d) Amino acid conservation alignment of five non‐synonymous variants identified in this study.

In family SS2, proband IV:2 was an 8‐year‐old boy who had progressive high myopia from the age of one (Figure 1b). Exome sequencing and co‐segregation analysis showed that the missense mutation c.1693C>T (p.Arg565Cys) in the COL2A1 gene was a pathogenic mutation in this family (Figure 1c). R565 was located on the collagen alpha‐1 chain of the COL2A1 protein (Figure 3c) and was at the X‐position of the G‐X‐Y triplet. This mutation has been previously reported (Wang et al., 2016; Zhou et al., 2018) and was predicted to be deleterious. The amino acids involved were highly conserved among vertebrates (Figure 3d and Table S2), and the penetrance of high myopia was complete. A detailed examination of the six carriers showed that II:4 and III:1 had vitreoretinal degeneration, cataracts, midfacial hypoplasia, sensorineural hearing loss, joint hypermobility, and early onset degenerative arthritis; III:3 had vitreoretinal degeneration, midfacial hypoplasia, and sensorineural hearing loss; and III:5 had vitreoretinal degeneration, cataracts, and midfacial hypoplasia. In addition, the II:4 group also showed spinal abnormalities (Tables S3 and S4).

In family SS3, the proband was a 23‐year‐old male with high myopia from the age of eight (Figure 2a). Genetic analysis revealed a missense mutation c.3505G>A (p.Val1169Ile) in COL2A1 in six subjects (Figure 2a). V1169 was located on the collagen alpha‐1 chain of the COL2A1 protein (Figure 3c) and was at the Y‐position of the G‐X‐Y triplet. This mutation was predicted to be deleterious, and the amino acids involved were highly conserved among many vertebrates (Figure 3d and Table S2). The c.3505G>A (p.Val1169Ile) mutation has been previously reported as a variant of COL2A1 protein (Clinvar database: VCV000881132.4), but its clinical significance is benign or uncertain. We further discovered that this mutation is pathogenic and causes STL1 through clinical examination, co‐segregation analysis and functional experiment, indicating that it is a novel STL1‐associated mutation. Half of the carriers had a high degree of myopia, and the rest showed slight myopia, which indicates an incomplete penetrance of this mutation. No other symptoms were observed in the six carriers. Detailed clinical information on high myopia is presented in Table S3.

In family SS4, proband IV:1 was a 22‐year‐old female with high myopia (Figure 2b). A genetic study revealed that the c.2895+1G>C mutation in intron 42 of COL2A1 was pathogenic (Figure 2b,c). This variation was absent in the GnomAD database and has not yet been reported yet. This mutation causes aberrant splicing and a 77 bp insertion into the coding sequence, leading to frameshifting, as revealed by the minigene assay (Figure 2d,e). In addition, c.2895+1G>C was located on the collagen alpha‐1 chain of the COL2A1 protein (Figure 3c) and between two G‐X‐Y triplets encoded by exons 42 and 43. The 77 bp insertion disrupted consecutive G‐X‐Y triplets (Figure 2e). After a detailed examination, we found that only IV:1 had a cleft palate, and no other symptoms were noticed in any of the five carriers. Detailed clinical information on high myopia is presented in Table S3.

3.2. Genetic studies revealed likely benign variants in COL2A1

Apart from the four STL1‐associated mutations, we identified two more rare variants on COL2A1, c.2806G>A (p.Gly936Ser) in family HM1 and c.550G>A (p.Ala184Thr) in family HM2, but they were not co‐segregated with high myopia phenotype in each family (Figure 3a and Table S2).

In family HM1, proband I:2 was a 77‐year‐old female with high myopia (OD: −13.80D, OS: −4.00D; Figure 3a). Exome sequencing identified a missense mutation, c.2806G>A (p.Gly936Ser), in COL2A1. The mutation c.2806G>A changed the glycine residue to serine in the G‐X‐Y triplet, and the interpretation in the ClinVar database was likely pathogenic (Clinvar database: VCV001066127.4; Figure 3c). In this family, only I:2 carried the mutation, whereas all other patients and normal subjects were wild types (Figure 3a). The mutation c.2806G>A was not co‐segregated with the phenotype in the family HM1. Further examination of I:2 did not show any STL1‐related abnormalities in the ophthalmology, auditory, midfacial, or musculoskeletal systems. The absence of STL1‐related abnormality in I:2 carrying a c.2806G>A mutation suggests that not all mutations that alter the glycine residue of the G‐X‐Y unit can cause STL1.

Proband III:2 of family HM2 was an 18‐year‐old male with high myopia (Figure 3b). The RE was −8.50D in the left eye and −9.50D in the right eye. Exome analysis identified a missense mutation, c.550G>A (p.Ala184Thr), in the COL2A1 gene of III:2. Mutation c.550G>A was located in the N‐terminal region of the collagen alpha‐1 chain (Figure 3c), and the interpretation in the ClinVar database was benign (VCV001053398.4). Sanger sequencing showed that the c.550G>A mutation was inherited from III:2's asymptomatic father, II:3. The mutation also did not co‐segregate with high myopia in the HM2 family (Figure 3b). In addition, there were no STL1‐related abnormalities in III:2. Thus, c.550G>A may be a polymorphism.

3.3. COL2A1 protein with STL1‐associated mutations tends to form aggregates and elevate ER stress

In HeLa cells transfected with plasmids encoding wild‐type (WT) COL2A1 or STL1‐associated mutants (R565C and V1169I), the distribution of WT COL2A1 showed no polarity and was either connected to form a network structure around the nucleus or appeared as small dots (net: 85.4 ± 3.9%, dots: 11.7 ± 3.0%, aggregates: 2.9 ± 1.1%; Figure 4a). However, the R565C mutants tended to form large perinuclear aggregates, and the proportion of cells with aggregates increased significantly (net: 61.8 ± 3.4%, dots: 21.2 ± 5.0%, aggregates: 17.1 ± 2.1%; Figure 4b,d). V1169I mutants occasionally appeared as aggregates but more frequently as discrete dots (net: 64.0 ± 5.4%, dots: 29.7 ± 4.6%, aggregates: 6.3 ± 2.5%; Figure 4c,d). All three forms of COL2A1 signals overlapped with the ER network (Figure 4e–g).

FIGURE 4.

FIGURE 4

Open in a new tab

Cellular distribution of COL2A1 mutants in HeLa cells. (a–c) HeLa cells were transiently transfected with flag‐tagged WT or mutant constructs. After 48 h, cells were fixed and stained with an anti‐flag antibody to reveal protein expression. Flag signals were classified into ‘net’, ‘dot’ and ‘aggregate’. Right panel is a higher magnification view of yellow boxes in middle panel. Cells transfected with WT COL2A1 were shown in (a), while R565C were in (b) and V1169I in (c). Scale bar: 10 μm. (d) Quantitative analysis of the percentage of cells with different types of COL2A1. Over 100 cells per group were analyzed in each experiment, and three independent experiments were performed. Data are presented as mean ± SEM. One‐way ANOVA followed by Dunnett's multiple comparison test. *p < 0.05, **p < 0.01. (e) COL2A‐flag was retained in the ER. COL2A1 WT was localized widely in the periphery as well as in the perinuclear region, as indicated by calnexin, an ER marker. Right panel is a higher magnification view of yellow boxes in left panel. Scale bar: 10 μm. (f, g) Both dots and aggregates form of R565C mutant located in ER. Right panel is a higher magnification view of yellow boxes in left panel. Scale bar: 10 μm.

The abnormal distribution of COL2A1 mutants imposes stress on the ER (Chung et al., 2009; Liang et al., 2014) and disturb intracellular homeostasis. Considering the central role of the PERK‐eIF2α‐ATF4 pathway, unfolded protein response to ER stress, phosphorylation level of PERK and protein level of ATF4 were determined. As shown in Figure 5a, R565C caused an increase in the levels of ATF4 and phosphor‐PERK (Figure 5a,b). the expression of the V1169I and R533* mutant did not elevate ATF4 levels significantly, but there was a rising trend in the phosphorylation level of PERK in these mutants group compared to WT (Figure 5a,b). Therefore, activation of the UPR indicated that the COL2A1 mutants imposed ER stress. Although the R565C mutation led to protein aggregation, it did not significantly induce autophagic flux (Figure 5c). The transition of LC3B‐I to LC3B‐II, a marker of autophagosome formation and maturation, remained the same in cells expressing WT, R565C, or V1165I COL2A1 (Figure 5c). Furthermore, cells expressing COL2A1 R565C mutant showed a steady increase of cleaved Caspase3, indicating a trend of apoptosis (Figure 5d,e).

FIGURE 5.

FIGURE 5

Open in a new tab

STL1‐associates mutations arouse ER stress and activate the unfolded protein response pathways. (a) PERK pathway in COL2A1 WT, R565C, V1169I, R533* expressed HeLa cells. HeLa cells were transfected with flag tagged COL2A1 WT, R565C, V1169I or R533* constructs. The level of ATF4, PERK and phosphor‐PERK (p‐PERK/PERK) was evaluated by western blotting. COL2A1 and its mutants were detected using an anti‐flag antibody. β‐Actin was used as a loading control. (b) Quantitative analysis of ATF4, PERK, phosphorylation of PERK presented in a. n = 4. One‐way ANOVA followed by Dunnett's multiple comparison test. *p < 0.05. Data are represented as mean ± SEM. (c) STL1‐associates mutations did not enhance autophagy. The transition from LC3B‐I to LC3B‐II were analyzed in cells with COL2A1 WT, R565C, V1169I or R533*. COL2A1 and its mutants were detected using anti‐flag antibody. β‐Actin was used as a loading control. (d) The cleaved Caspase 3 level was analyzed in cells expressing COL2A1 WT, R565C, V1169I or R533*. β‐Actin was used as a loading control. (e) Quantitative analysis of cleaved Caspase 3 presented in d. n = 3. One‐way ANOVA followed by Dunnett's multiple comparison test. *p < 0.05. Data are represented as mean ± SEM.

3.4. Retrospective genotype–phenotype correlation study of COL2A1 mutations

To comprehensively evaluate the genotype–phenotype correlation of STL1, we collected publications from 1989 to 2021 with detailed descriptions of COL2A1 mutations and clinical phenotypes. The correlation study included a total of 172 COL2A1 gene mutations in 830 patients, including four reported in this study (Dataset S1). As shown in Figure 6a, missense, nonsense, and frameshift indels were the predominant mutations in COL2A1. The mutations were ubiquitously distributed, and there were no hotspot clusters despite exons 2 and 42 having the maximum mutation number (Figure 6b). Viewed from protein structure, mutations were enriched in the collagen alpha‐1 chain region compared with the N‐ and C‐terminal regions (62.9 vs. 30.4 mutations/kb, p < 0.0001) (Figure 6b). The nonsense (16.4 vs. 7.8 mutations/kb, p = 0.0269) and missense (24.8 vs. 4.7 mutations/kb, p < 0.0001) mutations were predominantly attributed to this difference (Figure 6b). The most frequent mutations were c.1693C>T (p.Arg565Cys), c.1957C>T (p.Arg653*), and c.2794C>T (Arg932*) (Figure 6c).

FIGURE 6.

FIGURE 6

Open in a new tab

Genotype study of COL2A1 variants. (a) Pie plot of mutation types' distribution. (b) The number of mutations for each mutation type on the COL2A1 gene. E, exon. (c) The number of probands for each mutation type on the COL2A1 gene. E, exon.

Ophthalmologic abnormalities, such as high myopia, vitreous abnormality, and retinal detachment, were the leading phenotypes caused by the COL2A1 mutation (Figure 7a). At the same time, glaucoma, cleft palate, and hearing loss were minor symptoms. The predominant phenotypes caused by nonsense and frameshift indel mutations were high myopia, vitreous abnormalities, and retinal detachment (Figure 7a). Missense mutations constantly interrupt musculoskeletal symptoms, including hypermobile joints, arthritis, and spinal abnormalities (Figure 7a). Splicing site mutations mainly contribute to ophthalmologic‐related phenotypes such as high myopia, vitreous abnormalities, and facial‐related symptoms such as flattened face and micrognathia.

FIGURE 7.

FIGURE 7

Open in a new tab

Frequency of STL1 characteristics for COL2A1 gene mutations. (a) Frequency of STL1 characteristics for different mutation types. (b) Frequency of STL1 characteristics for mutations on collagen alpha‐1 chain and non‐alpha‐1 region. (c) Frequency of STL1 characteristics for mutations in exon 2 and others. E2, exon 2; HM, High myopia; VA, vitreoretinal abnormal; GC, Glaucoma; CT, Cataract; RD, Retinal detachment; FF, Flattened face; CP, Cleft palate; MG, Micrognathia; HL, Hearing loss; HJ, Hypermobile joints; ART, Arthritis; SA, Spinal abnormal; NS, No symptom. (d) Sanger sequencing results for mutation c.147G>A from two sporadic patients, M23298 and M30132. (e) Construction of minigene COL2A1 Exon 1–3 with c.147G>A mutation. Amplified RT‐PCR products were revealed. The transcript of wild‐type COL2A1 E1–3 is 464 nt.

Compared to the N‐ and C‐terminal sequences, mutations in the collagen alpha‐1 chain region contribute more to non‐ophthalmologic symptoms, including flattened face, cleft palate, micrognathia, hearing loss, hypermobile joint, and arthritis (Figure 7b). Retinal detachment is predominantly caused by the collagen non‐alpha‐1 chain region. In accordance, the four STL1‐associated mutations reported in this study all leading to non‐ophthalmologic phenotypes to some extent. Because of the frequent mutations in exon 2, we compared the phenotypes caused by exon 2 with those of other exons. Mutations in exon 2 mainly cause retinal detachment, while non‐ophthalmologic phenotypes (flattened face, cleft palate, micrognathia, hypermobile joints, arthritis, and spinal abnormalities) are primarily attributed to non‐exon 2 mutations (Figures 6b and 7c). Other proportions of ophthalmologic symptoms, including high myopia, vitreous abnormality, and cataracts, did not show significant difference between the alpha‐1 chain region and non‐alpha‐1 chain region, as well as exon 2 and other exons (Figure 7b,c). The discrepancy in the occurrence of ophthalmologic and non‐ophthalmologic phenotypes revealed by the genotype–phenotype correlation study paves the way for the mechanistic study of COL2A1 in STL1 and its precise treatment.

3.5. Rare COL2A1 mutations in exon 2 were identified from sporadic subjects with high myopia

Given that exons 2 haves the maximum mutation number and predominantly cause ophthalmologic symptoms (Figures 6b and 7c), we further scrutinized 200 sporadic patients with high myopia and revealed a rare synonymous mutation c.147G>A (p.Pro49Pro) in two subjects (M23298 and M30132) (Figure 7d). The allele frequency of c.147G>A in the gnomAD_exome database was 0.0001. M23298 was an 85‐year‐old female with RE −7.0D for the right and −6.0 for the left eye. Bilateral cataracts and a prolonged axial length were observed, with 28.61 mm for the right and 27.13 mm for the left eye. M30132 was a 37‐year‐old male with bilateral high myopia. The binocular RE and axial length were −16.0D and 32.80 mm, respectively. Moreover, biocular vitreous opacity and early onset cataracts were observed in the right eye.

The variant c.147G>A changed the adjacent sequence ‘AAGCCggAGCC’ to ‘AAGCCagAGCC’, creating a potential splicing acceptor. Therefore, we performed a minigene assay to evaluate the splicing effect of this variant. Electrophoresis and Sanger sequencing revealed identical band sizes and sequences between wild‐type and mutant, indicating a normal splicing event (Figure 7d). Whether the mutation is hazardous and whether it affects translation efficiency requires further investigation.

4. DISCUSSION

COL2A1 is predominantly expressed in collagen‐rich organs and tissues, including the vitreous, inner ear, and cartilage (Rukavina et al., 2014). Full‐length COL2A1 comprises a signal peptide, an N‐terminal pro‐peptide, a collagen alpha‐1 chain, and a C‐terminal pro‐peptide. During collagen maturation, three pro‐alpha‐1 chains encoded by COL2A1 tangle to form procollagen molecules, which are then processed into fibrils, followed by further crosslinking to form mature type II collagen fibrils (Robins, 2006). The G‐X‐Y triplet located in the collagen alpha‐1 chain plays a pivotal role in helix folding and stability (Engel & Bächinger, 2005). Sufficient native COL2A1 collagen is necessary for the stability of the ECM and function of cell (Arseni et al., 2018). Approximately 600 variants have been recorded in the HGMD database (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=COL2A1). Nonsense and frameshift mutations of COL2A1 are common in STL1 patients, indicating haploinsufficiency as a pathogenic mechanism (Barat‐Houari, Sarrabay, et al., 2016). This is mainly due to nonsense‐mediated mRNA decay or truncated proteins lacking the full length of the triple helix folding domain and the C‐terminal pro‐peptide necessary for helix folding, processing, and stability (Hoornaert et al., 2006). A few variants are missense mutations, and approximately 6% of STL1 patients are caused by dominant‐negative mutants that disrupt the G‐X‐Y triplet in the triple helix folding domain (Deng et al., 2016).

The nonsense mutation c.1597C>T (p.Arg533*) in family SS1 was located at the Y position of the G‐X‐Y triplet of the collagen alpha‐1 chain. The mutation may cause COL2A1 mRNA degradation through nonsense‐mediated decay (NMD) or result in a COL2A1 protein truncated with only one‐third of the full‐length collagen alpha‐1 chain. Since all plasmids encoding COL2A1 carried a flag tag fused at C‐terminal, COL2A1 R533* failed to express flag tag due to premature termination codon (Figure 5). Given that cells expressing COL2A1 R533* showed a trend of ER stress response, this truncated mutant could be expressed and accumulate in cells instead of nonsense‐mediated RNA decay. The mutation c.1597C>T has been reported previously (Liberfarb et al., 2003; Wilkin et al., 2000). In this study, we found that high myopia was the primary symptom in the SS1 family and reported, for the first time, that the c.1597C>T mutation was not fully penetrant. One of the explanations for the incomplete penetrance of this mutation may be that the nonsense mutation c.1597C>T lowers the functional COL2A1, resulting in a compensation effect to induce production of wild‐type collagen protein in heterozygous carriers. Unfortunately, we failed to test this hypothesis due to the lack of patient tissue or lymphocyte cell samples.

The missense mutation c.1693C>T is the most frequent variant of COL2A1, leading to STL1. In family SS2 with the c.1693C>T mutation, the penetrance for high myopia was complete. Other significant symptoms included vitreoretinal degeneration, cataracts, sensorineural hearing loss, and midfacial hypoplasia, as previously reported (Hoornaert et al., 2006; Richards et al., 2000; Wang et al., 2016; Zhou et al., 2018). The mutation c.1693C>T (p.Arg565Cys) was located at the X‐position of the G‐X‐Y triplet. Cysteine residues are generally absent in the COL2A1 triple‐helix domain (Kielty & Grant, 2002). The alternation of arginine to cysteine may give rise to a novel disulfide bond between mutated procollagen and disorganized alignments and trimer formation, thus restraining collagen in the rough ER and reducing the amount of intact mature collagen in the ECM (Barat‐Houari, Dumont, et al., 2016; Richards et al., 2002). Because this mutation may not induce compensation effect, the penetrance appears higher than that of the nonsense mutation c.1597C>T. Previous reports showed that mutated collagen was retained in the ER and caused ER stress, leading to apoptosis (Chung et al., 2009; Liang et al., 2014). Here, we noticed that the COL2A1 protein with Arg565Cys tended to form large aggregates in the ER, elevating ER stress and UPR in cells (Figure 5a,b). However, there were no signs of massive autophagy—level of LC3B‐II, and only a steady increase of apoptosis (Figure 5c–e). Since WT COL2A1 co‐existed with mutants in this cell model, mutant collagens may incorporate normal collagens to sustain function.

Here, we provide the first pathogenic evidence that the c.3505G>A (p. Val1169Ile) mutation correlates with STL1. Three out of six carriers showed high myopia, which indicates an incomplete penetrance. V1169I was located at the Y‐position of the type II collagen G‐X‐Y triplet. Valine and isoleucine are nonpolar neutral residues in the aliphatic class, with a similar hydropathy index (4.2 and 4.5, respectively). Therefore, alteration of valine to isoleucine may have little effect on the properties of the G‐X‐Y triplet. In addition, COL2A1 with the V1169I mutation forms dots in the ER and slightly increases phosphorylated PERK. Mild ER stress may explain the incomplete penetrance and slight phenotypes (only high myopia) for this mutation.

The missense mutations c.550G>A (p.Ala184Thr) and c.2806G>A (p.Gly936Ser) found in the HM2 and HM1 families did not co‐segregate with the high myopia phenotype. Carriers did not show STL1‐related symptoms such as vitreoretinal abnormalities, cataracts, midfacial hypoplasia, arthritis, hearing loss, or spinal changes, indicating that these mutations were not disease‐causing. The c.550G>A mutation was located on the N‐terminal region of the collagen alpha‐1 chain, and its interpretation in the ClinVar database was benign (https://www.ncbi.nlm.nih.gov/clinvar/variation/547247/). However, this site was not involved in the G‐X‐Y triplet, suggesting an inferior effect of mutations out of the G‐X‐Y unit. Although c.2806G>A changed the essential glycine residue to serine on the G‐X‐Y triplet, and the interpretation in the ClinVar database was likely pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/1066127/), it was not disease‐causing based on our study. Therefore, we concluded that mutating the glycine residue of the G‐X‐Y unit is not sufficient to cause STL1.

Among all the symptoms caused by COL2A1 gene mutations, the cardinal phenotypes are high myopia, vitreous abnormality, retinal detachment, and hypermobile joints. The incidence of glaucoma was the lowest in STL1 patients, and the majority of STL1 patients seeking treatment had ophthalmologic abnormalities. Because the combination of high myopia, cataracts, and retinal detachment commonly appears in various syndromic and non‐syndromic diseases, it is difficult to discriminate STL1 from other ophthalmologic‐related disorders based solely on routine ophthalmologic examination (Alsubaie et al., 2021; Čopíková et al., 2020; Du et al., 2021; Kjellstrom et al., 2021). Therefore, it is highly recommended that ophthalmologists pay more attention to non‐ophthalmologic symptoms like hypermobile joints, midfacial hypoplasia, arthritis, and spinal changes when inspecting a potential STL1 patient. Our study further strengthens that exome sequencing‐based genetic screening is a promising strategy to aid in identifying causal mutations. This greatly benefits the control of disease progression, precise intervention, genetic counseling, and even antenatal or preimplantation genetic testing.

5. CONCLUSIONS

In this study, we identified four STL1 families with COL2A1 mutations (c.1597C>T, c.1693C>T, c.2895+1G>C, and c.3505G>A) and two families with likely benign variants in COL2A1 (c.550G>A and c.2806G>A). Mechanistic study revealed that STL1‐related mutations showed abnormal cellular distribution and aggravated ER stress. Furthermore, genotype–phenotype correlation analysis was performed to elucidate the relationship between COL2A1 gene mutations and STL1 symptoms. This study expands the knowledge of COL2A1 gene mutations, highlights the importance of non‐ophthalmological examination in clinical diagnosis, emphasizes the necessity of exome sequencing for the precise diagnosis of ophthalmologic disorders, and provides evidence for further mechanistic studies of pathogenic mutations in the COL2A1 gene.

AUTHOR CONTRIBUTIONS

Qi Tian and Runyi Tian designed experiments. Runyi Tian and Ping Tong conducted experiments under the supervision of Qi Tian. Ping Tong conducted clinical evaluations of subjects. Qi Tian and Runyi Tian analyzed data and performed statistical analysis. Qi Tian and Runyi Tian wrote the manuscript. Yuhong He, Liyu Zang and Shimin Zhou assisted experiments. All authors have read and approved the article.

FUNDING INFORMATION

This study was supported by the Natural Science Foundation of Hunan Province (2021JJ40811) to Qi Tian, (2019JJ40408) to Shimin Zhou.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

Dataset S1.

Click here for additional data file. (80.1KB, docx)

Table S1.

Click here for additional data file. (36.8KB, docx)

ACKNOWLEDGMENTS

We would like to thank Dr. Xuemin Jin and Dr. Lei Tian from the First Affiliated Hospital of Zhengzhou University for their assistance with subject recruitment and sample collection. We would also like to thank Dr. Kun Xia and Dr. Zhengmao Hu, who kindly provided advice on this manuscript.

Tian, R. , Tong, P. , He, Y. , Zang, L. , Zhou, S. , & Tian, Q. (2024). Exome sequencing‐aided precise diagnosis of four families with type I Stickler syndrome. Molecular Genetics & Genomic Medicine, 12, e2331. 10.1002/mgg3.2331

Runyi Tian and Ping Tong contributed equally to this work.

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available in the article and supporting materials. Sequence variants have been submitted to the ClinVar database with accession number SCV002576562‐SCV002576568. (https://www.ncbi.nlm.nih.gov/clinvar/?gr=1&term=SUB12118374).

REFERENCES

  1. Alsubaie, H. F. , Magliyah, M. S. , AlRaddadi, O. , AlZaid, A. , & Nowilaty, S. R. (2021). Familial exudative vitreoretinopathy‐like features in Stickler type IV associated with novel variants in COL9A1. Retinal Cases and Brief Reports, 17, 206–211. 10.1097/ICB.0000000000001155 [DOI] [PubMed] [Google Scholar]
  2. Arseni, L. , Lombardi, A. , & Orioli, D. (2018). From structure to phenotype: Impact of collagen alterations on human health. International Journal of Molecular Sciences, 19(5), 1407. 10.3390/ijms19051407 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barat‐Houari, M. , Dumont, B. , Fabre, A. , Them, F. T. , Alembik, Y. , Alessandri, J. L. , Amiel, J. , Audebert, S. , Baumann‐Morel, C. , Blanchet, P. , Bieth, E. , Brechard, M. , Busa, T. , Calvas, P. , Capri, Y. , Cartault, F. , Chassaing, N. , Ciorca, V. , Coubes, C. , … Touitou, I. (2016). The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype. European Journal of Human Genetics, 24(7), 992–1000. 10.1038/ejhg.2015.250 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barat‐Houari, M. , Sarrabay, G. , Gatinois, V. , Fabre, A. , Dumont, B. , Genevieve, D. , & Touitou, I. (2016). Mutation update for COL2A1 gene variants associated with type II collagenopathies. Human Mutation, 37(1), 7–15. 10.1002/humu.22915 [DOI] [PubMed] [Google Scholar]
  5. Brodsky, B. , & Persikov, A. V. (2005). Molecular structure of the collagen triple helix. Advances in Protein Chemistry, 70, 301–339. 10.1016/S0065-3233(05)70009-7 [DOI] [PubMed] [Google Scholar]
  6. Chung, H. J. , Jensen, D. A. , Gawron, K. , Steplewski, A. , & Fertala, A. (2009). R992C (p.R1192C) substitution in collagen II alters the structure of mutant molecules and induces the unfolded protein response. Journal of Molecular Biology, 390(2), 306–318. 10.1016/j.jmb.2009.05.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Čopíková, J. , Paděrová, J. , Románková, V. , Havlovicová, M. , Balaščáková, M. , Zelinová, M. , Vejvalková, Š. , Simandlová, M. , Štěpánková, J. , Hořínová, V. , Kantorová, E. , Křečková, G. , Pospíšilová, J. , Boday, A. , Meszarosová, A. U. , Turnovec, M. , Votýpka, P. , Lišková, P. , & Kremlíková Pourová, R. (2020). Expanding the phenotype spectrum associated with pathogenic variants in the COL2A1 and COL11A1 genes. Annals of Human Genetics, 84(5), 380–392. 10.1111/ahg.12386 [DOI] [PubMed] [Google Scholar]
  8. Deng, H. , Huang, X. , & Yuan, L. (2016). Molecular genetics of the COL2A1‐related disorders. Mutation Research, Reviews in Mutation Research, 768, 1–13. 10.1016/j.mrrev.2016.02.003 [DOI] [PubMed] [Google Scholar]
  9. Donoso, L. A. , Edwards, A. O. , Frost, A. T. , Ritter, R., 3rd , Ahmad, N. , Vrabec, T. , Rogers, J. , Meyer, D. , & Parma, S. (2003). Clinical variability of Stickler syndrome: Role of exon 2 of the collagen COL2A1 gene. Survey of Ophthalmology, 48(2), 191–203. 10.1016/s0039-6257(02)00460-5 [DOI] [PubMed] [Google Scholar]
  10. Du, Q. , Zhang, D. , Zhuang, Y. , Xia, Q. , Wen, T. , & Jia, H. (2021). The molecular genetics of Marfan syndrome. International Journal of Medical Sciences, 18(13), 2752–2766. 10.7150/ijms.60685 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engel, J. , & Bächinger, H. P. (2005). Structure, stability and folding of the collagen triple helix. In Brinckmann J., Notbohm H., & Müller P. K. (Eds.), Collagen: Primer in structure, processing and assembly (pp. 7–33). Springer. 10.1007/b103818 [DOI] [Google Scholar]
  12. Flitcroft, D. I. , He, M. , Jonas, J. B. , Jong, M. , Naidoo, K. , Ohno‐Matsui, K. , Rahi, J. , Resnikoff, S. , Vitale, S. , & Yannuzzi, L. (2019). IMI—Defining and classifying myopia: A proposed set of standards for clinical and epidemiologic studies. Investigative Ophthalmology & Visual Science, 60(3), M20–M30. 10.1167/iovs.18-25957 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoornaert, K. P. , Dewinter, C. , Vereecke, I. , Beemer, F. A. , Courtens, W. , Fryer, A. , Fryssira, H. , Lees, M. , Müllner‐Eidenböck, A. , Rimoin, D. L. , Siderius, L. , Superti‐Furga, A. , Temple, K. , Willems, P. J. , Zankl, A. , Zweier, C. , de Paepe, A. , Coucke, P. , & Mortier, G. R. (2006). The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene. Journal of Medical Genetics, 43(5), 406–413. 10.1136/jmg.2005.035717 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoornaert, K. P. , Vereecke, I. , Dewinter, C. , Rosenberg, T. , Beemer, F. A. , Leroy, J. G. , Bendix, L. , Björck, E. , Bonduelle, M. , Boute, O. , Cormier‐Daire, V. , de Die‐Smulders, C. , Dieux‐Coeslier, A. , Dollfus, H. , Elting, M. , Green, A. , Guerci, V. I. , Hennekam, R. C. , Hilhorts‐Hofstee, Y. , … Mortier, G. R. (2010). Stickler syndrome caused by COL2A1 mutations: Genotype‐phenotype correlation in a series of 100 patients. European Journal of Human Genetics, 18(8), 872–880. 10.1038/ejhg.2010.23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kielty, C. M. , & Grant, M. E. (2002). The collagen family: Structure, assembly, and organization in the extracellular matrix. In Royce P. M., & Steinmann B. (Eds.), Connective tissue and its heritable disorders (pp. 159–221). Wiley‐Liss Inc. 10.1002/0471221929.ch2 [DOI] [Google Scholar]
  16. Kjellstrom, U. , Martell, S. , Brobeck, C. , & Andreasson, S. (2021). Autosomal recessive Stickler syndrome associated with homozygous mutations in the COL9A2 gene. Ophthalmic Genetics, 42(2), 161–169. 10.1080/13816810.2020.1861309 [DOI] [PubMed] [Google Scholar]
  17. Liang, G. , Lian, C. , Huang, D. , Gao, W. , Liang, A. , Peng, Y. , Ye, W. , Wu, Z. , Su, P. , & Huang, D. (2014). Endoplasmic reticulum stress‐unfolding protein response‐apoptosis cascade causes chondrodysplasia in a col2a1 p.Gly1170Ser mutated mouse model. PLoS ONE, 9(1), e86894. 10.1371/journal.pone.0086894 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liberfarb, R. M. , Levy, H. P. , Rose, P. S. , Wilkin, D. J. , Davis, J. , Balog, J. Z. , Griffith, A. J. , Szymko‐Bennett, Y. M. , Johnston, J. J. , Francomano, C. A. , Tsilou, E. , & Rubin, B. I. (2003). The Stickler syndrome: Genotype/phenotype correlation in 10 families with Stickler syndrome resulting from seven mutations in the type II collagen gene locus COL2A1. Genetics in Medicine, 5(1), 21–27. 10.1097/00125817-200301000-00004 [DOI] [PubMed] [Google Scholar]
  19. McAlinden, A. , Majava, M. , Bishop, P. N. , Perveen, R. , Black, G. C. , Pierpont, M. E. , Ala‐Kokko, L. , & Männikkö, M. (2008). Missense and nonsense mutations in the alternatively‐spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome. Human Mutation, 29(1), 83–90. 10.1002/humu.20603 [DOI] [PubMed] [Google Scholar]
  20. Reardon, A. , Sandell, L. , Jones, C. J. , McLeod, D. , & Bishop, P. N. (2000). Localization of pN‐type IIA procollagen on adult bovine vitreous collagen fibrils. Matrix Biology, 19(2), 169–173. 10.1016/s0945-053x(00)00058-5 [DOI] [PubMed] [Google Scholar]
  21. Richards, A. J. , Baguley, D. M. , Yates, J. R. , Lane, C. , Nicol, M. , Harper, P. S. , Scott, J. D. , & Snead, M. P. (2000). Variation in the vitreous phenotype of Stickler syndrome can be caused by different amino acid substitutions in the X position of the type II collagen Gly‐X‐Y triple helix. American Journal of Human Genetics, 67(5), 1083–1094. 10.1016/S0002-9297(07)62938-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richards, A. J. , McNinch, A. , Martin, H. , Oakhill, K. , Rai, H. , Waller, S. , Treacy, B. , Whittaker, J. , Meredith, S. , Poulson, A. , & Snead, M. P. (2010). Stickler syndrome and the vitreous phenotype: Mutations in COL2A1 and COL11A1. Human Mutation, 31(6), E1461–E1471. 10.1002/humu.21257 [DOI] [PubMed] [Google Scholar]
  23. Richards, A. J. , Morgan, J. , Bearcroft, P. W. , Pickering, E. , Owen, M. J. , Holmans, P. , Williams, N. , Tysoe, C. , Pope, F. M. , Snead, M. P. , & Hughes, H. (2002). Vitreoretinopathy with phalangeal epiphyseal dysplasia, a type II collagenopathy resulting from a novel mutation in the C‐propeptide region of the molecule. Journal of Medical Genetics, 39(9), 661–665. 10.1136/jmg.39.9.661 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robin, N. H. , Moran, R. T. , & Ala‐Kokko, L. (1993). Stickler syndrome. In Adam M. P., Ardinger H. H., Pagon R. A., Wallace S. E., Bean L. J. H., Mirzaa G., & Amemiya A. (Eds.), GeneReviews is a registered trademark of the University of Washington. University of Washington. [Google Scholar]
  25. Robins, S. P. (2006). Fibrillogenesis and maturation of collagens. Dynamics of Bone and Cartilage Metabolism, 2, 41–53. [Google Scholar]
  26. Rukavina, I. , Mortier, G. , Van Laer, L. , Frkovic, M. , Dapic, T. , & Jelusic, M. (2014). Mutation in the type II collagen gene (COL2AI) as a cause of primary osteoarthritis associated with mild spondyloepiphyseal involvement. Seminars in Arthritis and Rheumatism, 44(1), 101–104. 10.1016/j.semarthrit.2014.03.003 [DOI] [PubMed] [Google Scholar]
  27. Sandell, L. J. , Nalin, A. M. , & Reife, R. A. (1994). Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non‐cartilaginous tissues during early mouse development. Developmental Dynamics, 199(2), 129–140. 10.1002/aja.1001990206 [DOI] [PubMed] [Google Scholar]
  28. Snead, M. P. , McNinch, A. M. , Poulson, A. V. , Bearcroft, P. , Silverman, B. , Gomersall, P. , Parfect, V. , & Richards, A. J. (2011). Stickler syndrome, ocular‐only variants and a key diagnostic role for the ophthalmologist. Eye (London, England), 25(11), 1389–1400. 10.1038/eye.2011.201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Snead, M. P. , & Yates, J. R. (1999). Clinical and molecular genetics of Stickler syndrome. Journal of Medical Genetics, 36(5), 353–359. http://www.ncbi.nlm.nih.gov/pubmed/10353778 [PMC free article] [PubMed] [Google Scholar]
  30. Stickler, G. B. , Belau, P. G. , Farrell, F. J. , Jones, J. D. , Pugh, D. G. , Steinberg, A. G. , & Ward, L. E. (1965). Hereditary progressive arthro‐ophthalmopathy. Mayo Clinic Proceedings, 40, 433–455. http://www.ncbi.nlm.nih.gov/pubmed/14299791 [PubMed] [Google Scholar]
  31. Stickler, G. B. , Hughes, W. , & Houchin, P. (2001). Clinical features of hereditary progressive arthro‐ophthalmopathy (Stickler syndrome): A survey. Genetics in Medicine, 3(3), 192–196. 10.1097/00125817-200105000-00008 [DOI] [PubMed] [Google Scholar]
  32. Tian, Q. , Li, Y. , Kousar, R. , Guo, H. , Peng, F. , Zheng, Y. , Yang, X. , Long, Z. , Tian, R. , Xia, K. , Lin, H. , & Pan, Q. (2017). A novel NHS mutation causes Nance‐Horan syndrome in a Chinese family. BMC Medical Genetics, 18(1), 2. 10.1186/s12881-016-0360-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang, X. , Jia, X. , Xiao, X. , Li, S. , Li, J. , Li, Y. , Wei, Y. , Liang, X. , & Guo, X. (2016). Mutation survey and genotype‐phenotype analysis of COL2A1 and COL11A1 genes in 16 Chinese patients with Stickler syndrome. Molecular Vision, 22, 697–704. http://www.ncbi.nlm.nih.gov/pubmed/27390512 [PMC free article] [PubMed] [Google Scholar]
  34. Wilkin, D. J. , Liberfarb, R. , Davis, J. , Levy, H. P. , Cole, W. G. , Francomano, C. A. , & Cohn, D. H. (2000). Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: Analysis of potential premature termination codons. American Journal of Medical Genetics, 94(2), 141–148. [DOI] [PubMed] [Google Scholar]
  35. Zhou, L. , Xiao, X. , Li, S. , Jia, X. , Wang, P. , Sun, W. , Zhang, Q. , Li, J. , Li, T. , & Zhang, Q. (2018). Phenotypic characterization of patients with early‐onset high myopia due to mutations in COL2A1 or COL11A1: Why not Stickler syndrome? Molecular Vision, 24, 560–573. [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Dataset S1.

Click here for additional data file. (80.1KB, docx)

Table S1.

Click here for additional data file. (36.8KB, docx)

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available in the article and supporting materials. Sequence variants have been submitted to the ClinVar database with accession number SCV002576562‐SCV002576568. (https://www.ncbi.nlm.nih.gov/clinvar/?gr=1&term=SUB12118374).

Articles from Molecular Genetics & Genomic Medicine are provided here courtesy of Blackwell Publishing

RESOURCES