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Introduction

Osteogenesis imperfecta (OI), also known as brittle bone disorder, is a connective tissue disorder caused by an abnormal structure and insufficient quantity of type I collagen or incorrect modification and folding after translation (1,2). OI affects ~1 in 15,000-20,000 individuals worldwide (3). The main clinical features of OI include skeletal fragility, mild injury, multiple nontraumatic fractures, skeletal deformities, blue/grey sclera, dentin hypoplasia, progressive hearing loss in adults and short stature (4). OI is characterized by strong genetic heterogeneity and a wide range of clinical phenotypic variations (3). Based on the different clinical manifestations, genetic bases and genetic patterns, OI is currently classified into ≥23 subtypes, of which the OI–I-IV subtypes account for >90% of cases and are directly caused by pathogenic variants in the COL1A1 and COL1A2 genes, with COL1A1 variants being the most common (5,6).

The COL1A1 gene is located at 17q21.33 and has a total length of 17,530 bp, containing 51 exons and 50 introns (7). The human COL1A1 gene has been identified to produce a transcript with an mRNA length of 4,395 bp (NM:000088), encoding the type I collagen α1 strand [pro α (I); NP_000079.2]. Thousands of pathogenic variants in COL1A1, including missense, nonsense, insertion, deletion, duplication and splice-site variants, have been reported. The frequency of splice-site variants is relatively high, accounting for ~15% of all recorded variants (5).

The present study used whole-exome sequencing (WES) technology to identify a heterozygous variant, c.3531+1G>T, at the splicing site in intron 47 of COL1A1 in a family with OI. Essawi et al (8) first reported this variant in the Palestinian population. However, to the best of our knowledge, no functional studies have been conducted on this variant to date. By extracting RNA from lymphocyte strains and performing reverse transcription (RT)-PCR, as well as conducting minigene experiments, the present study aimed to investigate whether this variant affects splicing. To the best of our knowledge, this is the first study on the effect of the c.3531+1G>T variant in the COL1A1 gene.

Materials and methods

Compliance with ethical standards

The present study fully complied with the tenets of The Declaration of Helsinki and was approved by the Ethics Board of the Women and Children's Hospital, Xiamen University, China (approval no. KY-2022-090-K02; Xiamen, China). Written informed consent to participate in the present study was provided by the participants, legal guardians or next of kin (when the participants were minors <18 years old or elderly participants).

Subjects and clinical description

In the family investigated, six affected individuals had a history of recurrent fractures without any other complications and four individuals were unaffected, as shown in the family pedigree (Fig. 1A). The proband (II:3) was born with a light blue sclera and fell at the age of 12 years, resulting in a fracture at the lower end of the right elbow bone. The proband had no other fractures since then. The proband was 34 years old and 156 cm tall. One healthy, age-matched female from a non-related family participated in the current study.

Figure 1. (A) Family pedigree. The arrow indicates that II:3 was the proband. I:2, II:1, II:2, III:2 and III:5 were other affected individuals in the family. (B) Sanger sequencing validation results of the affected individuals. All affected individuals in the family carried variant c.3531 in the COL1A1 gene. (C) Sanger sequencing validation results of the unaffected individuals. All the four unaffected individuals (I:1, III:1, III:3 and III:4) did not carry the variant.

WES

A QIAamp Blood Mini Kit (Qiagen GmbH) was used to extract DNA from the peripheral venous blood of the proband. Subsequently, the genomic DNA concentration was determined on Qubit 3.0 (cat. no. Q33216; Thermo Fisher Scientific, Inc.) using the Qubit dsDNA BR Assay Kit 100 assays (cat. no. Q32850; Thermo Fisher Scientific, Inc.). The concentration of genomic DNA was 131 ng/µl. In addition, 1% agarose gel electrophoresis was used to detect genomic DNA integrity. Subsequently, the genomic DNA was randomly fragmented using an ultrasonicator (cat. no. LE220; Covaris, Ltd.) for 130 sec (2×65 sec) at a default temperature, duty cycle of 30%, 450 peak incident power (W) and 200 cycles per burst in a 96-well microTUBE Plate. The temperature of the external cooler was below 10°C and 200–300 bp fragments were selected. Using the SureSelectXT Reagent Kit (cat. no. G9611B; Agilent Technologies, Inc.), Agilent V6 probe (cat. no. 5190-8863; Agilent Technologies, Inc.) and Agencourt® AMPure® XP Beads (cat. no. A63881; Beckman Coulter, Inc.), according to the manufacturers' protocols, the target gene exon library was prepared. Detected using Qubit 3.0 and the ssDNA Assay Kit (cat. no. 12645ES76; Shanghai Yeasen Biotechnology Co., Ltd.), the final library loading concentration was 13.77 ng/µl. Finally, paired-end 150-bp sequencing was performed on the MGISEQ-200RS platform (MGI Tech Co., Ltd.) using the MGISEQ-200RS High Throughput (Rapid) Sequencing Kit (cat. no. 1000012555; MGITech Co., Ltd.). The quality control indices for sequencing data were as follows: Average sequencing depth of the target area of ≥150X; and proportion of sites with an average depth of >20X in the target area >95%.

The sequenced fragments were compared with the UCSC hg19 human reference genome using Burrows-Wheeler Aligner [BWA-0.7.17 (r1188)] software (https://bio-bwa.sourceforge.net/) to remove duplicates. The Genome Analysis Toolkit software (https://software.broadinstitute.org/gatk/) was used for base quality correction and single nucleotide variant, insertion/deletion and genotype detection. ExomeDepth (1.1.15) (9) was used to detect copy number variants at the exon level.

Detected variants were filtered according to the following criteria: i) Frequency <1% according to the dbSNP (https://www.ncbi.nlm.nih.gov/snp/), 1000 Genomes Project (Phase3, http://www.internationalgenome.org/), ESP6500 (V2; http://genome.sph.umich.edu/wiki/NHLBI_Exome_Sequencing_Project), and gnomAD databases (r2.1.1; http://gnomad-sg.org/); ii) amino acid alterations and canonical splice site alterations; iii) homozygous, heterozygous or compound heterozygous variants; iv) amino acid conservation across species and pathogenicity prediction using in silico tools [VarSome (https://varsome.com/), CADD (https://cadd.gs.washington.edu/snv), MutationTaster (https://www.mutationtaster.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://provean.jcvi.org/seq_submit.php)]; v) genotype-phenotype analysis according to Exomiser (https://exomiser.readthedocs.io/) and Phenolyzer (https://phenolyzer.wglab.org/); vi) sequence variants interpreted following the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) variant-interpretation guidelines and the work groups from ClinGen (10–12). In addition, records were checked for in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) and the pathogenicity of splicing variants was predicted using VarCards2 (http://www.genemed.tech//varcards2/) (13).

Sanger sequencing

A QIAamp Blood Mini Kit (Qiagen GmbH) was used to extract DNA from the peripheral venous blood of all the family members. Primers designed using Oligo 6 (Molecular Biology Insights, Inc.) were used to amplify fragments of the candidate variant site of COL1A1 for the proband and all the family members, and an ABI 3130×l gene analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for forward and reverse sequencing.

Sequence analysis of COL1A1 cDNA

The present study established lymphocyte strains of the peripheral blood of the proband, the father (I:1) and the normal age-matched control according to the method introduced by Neitzel (14). Total RNA was extracted from the lymphocyte strains using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's recommended protocol. Subsequently, RNA was reverse transcribed to cDNA using the RT kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The COL1A1 cDNA was then amplified using two pairs of primers. The forward primer in the first pair of primers was located in exon 45, whereas the reverse primer was located in exon 48 (1F: 5′-TAAAGGGTCACCGTGGCTTCT-3′, 1R: 5′-CAGGCTCTTGAGGGTGGTGT-3′). The forward primer in the second pair of primers was located in exon 46, whereas the reverse primer was located in exon 49 (2F: 5′-CAAGGTCCCTCTGGAGCCTC-3′, 2R: 5′-GGTTGGGGTCAATCCAGTACTCT-3′). The amplification was carried out using the TaKaRa LA Taq® with GC Buffer (cat. no. RR02AG; Takara Biotechnology Co., Ltd.). The reaction conditions were as follows: Pre-denaturation at 94°C for 1 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 63°C for 30 sec and extension at 72°C for 45 sec; and a final extension at 72°C for 10 min and storage at 4°C. The PCR products were separated by polyacrylamide gel electrophoresis on 6% gels and were visualized with silver nitrate, and were then were separated using 2% agarose gels. The gels were then cut, purified and Sanger sequenced.

Minigene splicing assay

To verify the alternative splicing events, a minigene splicing assay was performed. The 293T cell line was selected to eliminate endogenous interference due to low expression of type I collagen. 293T cells (cat. no. CL-0005; Procell Life Science & Technology Co., Ltd.) were cultured at 37°C with 5% CO2 in DMEM (cat. no. PM150210; Procell Life Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (cat. no. 10091148; Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin (cat. no. SV30010; HyClone; Cytiva).

Wild-type hCOL1A1-Exon47-Exon48+c.3531+1G (hCOL1A1-W) and mutant-type hCOL1A1-Exon47-Exon48+ c.3531+1T (hCOL1A1-M) plasmids were constructed using the pSPL3b[minigene]-Puro-SV40 vector by Suzhou Haixing Biosciences Co., Ltd. Variants in minigene artefacts were confirmed using Sanger sequencing. Minigene plasmids were transfected using an electroporator (Neon™ Transfection System; cat. no. MPK5000; Thermo Fisher Scientific, Inc.), Neon Transfection System Pipette (cat. no. MPP100; Thermo Fisher Scientific, Inc.) and Neon Transfection System 100 µl Kit (cat. no. MPK10096; Thermo Fisher Scientific, Inc.) with 2×106 cells/100 µl. The electrical conversion parameters were as follows: Pulse temperature, ~23°C; Pulse voltage, 1,100 v; pulse duration, 20 msec; pulse number or pulse repetition frequency, 2. The EGFP plasmid (control plasmid) was electro-converted using the same parameters to determine the electrical conversion efficiency. After electroporation, the cells were harvested after immediately culturing at 37°C with 5% CO2 in pre-warmed DMEM supplemented with 10% fetal bovine serum for 48 h. Cells were processed for total RNA isolation using TRIzol and treated with RQ1 DNase (cat. no. M6101; Promega Corporation) to remove DNA according to the manufacturer's protocols. cDNA was synthesized using a RT kit (cat. no. R323-01; Vazyme Biotech Co., Ltd.), according to the manufacturer's protocol. Subsequently, PCR was performed to verify alternative events. The primers used for amplifying fragments containing this site were as follows: pSPL3b-SD6: 5′-TCTGAGTCACCTGGACAACC-3′; pSPL3b-SA2: 5′-ATCTCAGTGGTATTTGTGAGC-3′. The amplification was carried out using the 2X Taq Master Mix (Dye Plus) (cat. no. P112; Vazyme Biotech Co., Ltd.). The reaction conditions were as follows: Pre-denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 45 sec; and a final extension at 72°C for 10 min and storage at 4°C. The target product was 652 bp in length. Subsequently, the product was separated by electrophoresis on a 1.5% agarose gel, followed by ethidium bromide staining, cutting, purification. Finally, according to the manufacturer's protocols of the Ultra-Universal TOPO cloning kit (cat. no. C603; Vazyme Biotech Co., Ltd.), TA cloning was performed and the purified fragment underwent Sanger sequencing.

Results

Identification of candidate variants via WES and Sanger sequencing

High-quality sequencing data were obtained via WES. For the proband, 15 Gb data were generated with a 99.78% coverage of the target region and an average sequencing depth of 144 reads. After filtering and analysis, a heterozygous splice site variant (c.3531+1G>T) in the COL1A1 gene was found in the proband.

Sanger sequencing confirmed the presence of heterozygous variants in the proband. In addition, all of the other five affected individuals in the family (Fig. 1B) and none of the four unaffected individuals carried the variant (Fig. 1C).

There were no records of this variant in the dbSNP, 1000 Genomes Project, ESP6500 and ExAC databases. ClinVar includes this variant and annotates it as a pathogenic variant. The dbscSNV ADA SCORE was 1 and dbscSNV RF SCORE was 0.906 from VarCards2. However, to the best of our knowledge, no functional studies have been conducted on this variant to date. Therefore further analyses were conducted to assess its role in OI.

Sequence analysis of COL1A1 cDNA

COL1A1 cDNA samples of the lymphocyte strains from both the proband and normal age-matched control were compared. The target product (col1a1-V1) had lengths of 395 and 454 bp when amplified using the first (1F1R) and second (2F2R) primer pairs, respectively. Both the proband and the healthy individual had an additional product (col1a1-V2) that was slightly longer than the target product (~490 or 550 bp; Fig. 2A). In the healthy individual, the expression of col1a1-V1 was high, whereas that of col1a1-V2 was low. By contrast, the proband showed low expression of col1a1-V1 and high expression of col1a1-V2. In-depth Sanger sequencing of these bands revealed that col1a1-V1 did not contain intron 47 (Fig. 2B), while the slightly longer col1a1-V2 retained it (Fig. 2B).

Figure 2. (A) Electrophoresis results of human cDNA. When the 1F1R and 2F2R primer pairs were used to amplify normal human COL1A1 cDNA, target products (col1a1-V1) of 395 and 454 bp in length were produced, respectively. However, the normal individual and the proband had an additional band (col1a1-V2) (~490 and 550 bp, respectively) that was slightly longer than the target band. In normal individuals, the expression of col1a1-V1 was high, whereas that of col1a1-V2 was low. By contrast, the proband showed low expression of col1a1-V1 and high expression of col1a1-V2. (B) Sanger sequencing of human cDNA. Col1a1-V1 of both normal individual and the proband did not contain intron 47. Col1a1-V2 retained intron 47. N, normal individual; P, proband.

Minigene identification

The construction of a minigene plasmid is shown in Fig. 3A. Minigene splicing assays showed that plasmid hCOL1A1-W, underwent three types of splicing (col1a1-V1, V2 and V3; Fig. 3B and C). Col1a1-V1 was a result of normal splicing. Col1a1-V2 retained the entire intron 47, with the first base being G. Col1a1-V3 removed the entire intron 47 and partially removed exon 48, leading to deletion of the sequence from nucleotide c.3532 to c.3603 (c.3532_3603del). The agarose gel electrophoresis bands corresponding to col1a1-V1 and V3 from hCOL1A1-W were very light and cannot be observed in Fig. 3C. The sequencing results have been detailed in the Supplementary Materials (Fig. S1).

Figure 3. (A) Vector diagram. Plasmids were prepared according to the diagram. The W and M plasmids both contained the human intron 47 sequence, but the W plasmid had a G, while the M plasmid had a T at position c.3531+1. pSPL3b-SD6 referred to the forward primer used in PCR and pSPL3b-SA2 to the reverse primer. (B) W exhibited three splicing patterns: Col1a1-V1, col1a1-V2 and col1a1-V3. In col1a1-V1, the intron 47 was removed. Col1a1-V2 retained the entire intron 47. In col1a1-V3 the entire intron 47 and part of exon 48 was deleted. The red arrow represents the position of c.3531+1, where the base is a wild-type G. The black arrow represents the position of the splicing. (C) Electrophoresis results of the minigene splicing assays. The arrows indicates the positions of the bands corresponding to col1a1-V1, col1a1-V2 and col1a1-V4. The V2 and V3 bands from hCOL1A1-W cannot be observed, and the V5 and V6 bands from hCOL1A1-M cannot be observed. (D) M exhibited four splicing patterns, i.e. col1a1-V2, col1a1-V4, col1a1-V5 and col1a1-V6. Col1a1-V2 retained the entire intron 47. In col1a1-V4 the entire exon 47 and intron 47 was deleted. Col1a1-V5 retained part of intron 47 and part of exon 48. Col1a1-V6 retained the entire intron 47 and part of exon 48. The red arrow represents the position of c.3531+1, where the base is a mutant-type T. The black arrow represents the position of the splicing. W, wild type plasmid hCOL1A1-W; M, mutation type plasmid hCOL1A1-M; NC, blank control (water was used as a template).

By contrast, plasmid hCOL1A1-M underwent four types of splicing (col1a1-V2, V4, V5 and V6; Fig. 3C and D). Col1a1-V2, like wild-type COL1A1, retained intron 47, with the first base being T. In col1a1-V4, the entire exon 47 and intron 47 were removed. Col1a1-V5 partially retained intron 47 and exon 48, leading to the insertion of the intron 47 sequence from positions c.3531+1 to c.3531+30 [r.3531_3532ins(u; 3531+2_3531+30)] and the deletion of the sequence from nucleotides c.3532 to c.3699 (c.3532_3699del). Col1a1-V6 retained the entire intron 47 and partly retained exon 48, leading to the deletion of the sequence from nucleotides c.3674 to c.3773 (c.3674_3773del). The agarose gel electrophoresis bands corresponding to col1a1-V5 and V6 from hCOL1A1-M were very light and cannot be observed in Fig. 3C. These sequencing results have been detailed in Fig. S2.

Discussion

To date, 21 genes have been reported to cause OI (Table I), most of which lead to type I collagen synthesis disorders (15–19). Type I collagen is the main structural protein of bones and connective tissue, and is an important component that interacts with cell surfaces and the extracellular matrix. The triple helix structure of type I procollagen molecules consists of two α1 chains and one α2 chain, encoded by the COL1A1 and COL1A2 genes, respectively (20). Variants in these genes cause types I–IV OI. Extensive experimental and clinical observational studies have shown an association between clinical manifestations of OI and the sites of genetic variants. Since assembly of the triple helix starts from the C-terminus, variants in this region of the α1 and α2 chains can lead to more unstable collagen structures and more severe phenotypes (21,22). The type I phenotype of OI is relatively mild and is mainly caused by nonsense, frameshift or splice-site variants in COL1A1 and COL1A2, leading to premature translation termination, halving type I collagen synthesis (23). Types II–IV OI constitute more severe phenotypes; these cases are mainly caused by missense variants leading to amino acid substitutions, resulting in structural changes in type I collagen (22–25). Few pathogenic variants are caused by splicing variants, insertions, deletions or duplications, all of which may result in changes in the coding box sequence and carboxyl end peptide, thereby altering the structure of type I collagen (22). The proband and their affected family members in the present study were classified as having type I OI due to their mild phenotype.

Table I. Genes associated with osteogenesis imperfecta.

Table I.

Genes associated with osteogenesis imperfecta.

Gene	Locus	Gene OMIM number	Inheritance	OI type	Phenotype OMIM number
COL1A1	17q21.33	120150	AD	OI I	166200
COL1A1	17q21.33	120150	AD	OI II	166210
COL1A1	17q21.33	120150	AD	OI III	259420
COL1A1	17q21.33	120150	AD	OI IV	166220
COL1A2	7q21.3	120160	AD	OI II	166210
COL1A2	7q21.3	120160	AD	OI III	259420
COL1A2	7q21.3	120160	AD	OI IV	166220
IFITM5	11p15.5	614757	AD	OI V	610967
SERPINF1	17p13.3	172860	AR	OI VI	613982
CRTAP	3p22.3	605497	AR	OI VII	610682
P3H1	1p34.2	610339	AR	OI VIII	610915
PPIB	15q22.31	123841	AR	OI IX	259440
SERPINH1	11q13.5	600943	AR	OI X	613848
FKBP10	17q21.2	607063	AR	OI XI	610968
SP7	12q13.13	606633	AR	OI XII	613849
BMP1	8p21.3	112264	AR	OI XIII	614856
TMEM38B	9q31.2	611236	AR	OI XIV	615066
WNT1	12q13.12	164820	AR	OI XV	615220
CREB3L1	11p11.2	616215	AR	OI XVI	616229
SPARC	5q33.1	182120	AR	OI XVII	616507
TENT5A	6q14.1	611357	AR	OI XVIII	617952
MBTPS2	Xp22.12	300294	XLR	OI XIX	301014
MESD	15q25.1	607783	AR	OI XX	618644
KDELR2	7p22.1	609024	AR	OI XXI	619131
CCDC134	22q13.2	618788	AR	OI XXII	619795
PHLDB1	11q23.3	612834	AR	OI XXIII	620639

[i] AD, autosomal dominant; AR, autosomal recessive; OMIM, Online Mendelian Inheritance in Man; XLR, X-linked recessive.

Although the proband in the current study had only one fracture at the age of 12 years old, with normal height, a pale blue sclera, and no bone deformities or other complications, other patients in the family had recurrent fractures (the number of fractures was unknown). A patient with the same variant reported by Willing et al (26) had mild symptoms. However, Essawi et al (8) reported that a patient with OI caused by this variant had already suffered 15 fractures before the age of 5 years, and the patient had a short stature and blue sclera. Therefore, the OI phenotype caused by this variant is variable, ranging from mild to moderate.

Using WES and Sanger sequencing, the present study reported a heterozygous splice variant, c.3531+1G>T, in COL1A1 in patients from a family with OI. The results showed that this variant resulted in a decrease in the normal transcript (col1a1-V1) production and an increase in the abnormal transcript (col1a1-V2) production. In col1a1-V2, intron 47 was transformed into a coding sequence, resulting in a mutation of amino acid 1,178 from glycine to valine and the premature generation of a stop codon at position 92 from this site, which might lead to protein truncation (p.Gly1178Valfs*91). Multiple frameshift mutations have been reported downstream of this site, which would lead to truncated proteins and cause OI–I, such as c.3540del (p.Gly1181fs), c.3540dup (p.Gly1181fs), c.3566del (p.Pro1189fs), c.3566dup (p.Gly1190fs) and c.3567del (p.Gly1190fs) (26,27–32). Variants causing haploinsufficiency of COL1A1 are considered pathogenic (26,31,32). The present study further investigated splicing patterns using the minigene assay. The minigene experiment showed that hCOL1A1-M underwent four types of splicing, while hCOL1A1-W only underwent three types of splicing. Col1a1-V3, with a c.3532_3603del, resulting in amino acid deletions from positions 1,178-1,201 (p.Gly1178_Leu1201del), would miss five repeats of GXY in pro α (I). In col1a1-V4, the entire exon 47 and intron 47 were removed, resulting in amino acid deletions from positions 1,141-1,177 (p.Arg1141_Val1177del), leading to 12 repeats of GXY being missed in pro α (I). Col1a1-V5 retained part of intron 47 and part of exon 48, which resulted in an increase of 10 amino acids and a lack of five repeats of GXY in pro α (I). Col1a1-V6 retained the entire intron 47 and partially retained exon 48, resulting in the premature generation of a stop codon at the 81st amino acid after p.Val1177, forming a truncated protein. The reduction of GXY repeats in Col1a1-V3, V4 and V5 could affect the formation of the triple helix of collagen. These results indicated that the c.3531+1G>T variant had an effect on intron 47 splicing. The splice variant c.3531+1G>T contributed to the disappearance of the classic donor splice sites of intron 47, which led to intron 47 retention or partial retention.

Several previously identified variants at this splicing site, namely c.3531+1G>A, c.3531+1G>C, c.3531+1G>T and c.3531+2T>C, have been reported to lead to OI (6,8,26,31–34). Of these, experimental studies have revealed that the c.3531+1G>A variant leads to the skipping of exon 48 and reduced mRNA levels (26). No such experiments were conducted on the remaining variants. Previous studies have revealed other COL1A1 variants that may cause premature termination, leading to reduced mRNA stability in the mutated alleles (26,31,35). Ultimately, such a change would decrease type I collagen synthesis, resulting in type I OI. Based on the ACMG and AMP guidelines for interpreting sequence variants, this variant was classified as pathogenic. Specific evidence for the pathogenicity of this variant is as follows: i) PVS1: Null variant (canonical +/- 1 splice sites) in a gene where the loss of function is a known mechanism of disease; ii) PS2: In vivo and in vitro experiments in the present study suggested that this variant may affect the normal splicing of RNA; iii) PP1: It was consistent with co-segregation; this variant was detected in all six affected individuals; iv) PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product.

Notably, it was found that healthy individual also expressed the col1a1-V2 transcript but at markedly lower level than it in the proband. A minigene experiment also confirmed the presence of col1a1-V2 as well as a transcript that produces a truncated exon 48 from the wild-type gene. These results suggested that the normal COL1A1 gene might generate alternative transcripts. The minigene experiment revealed more transcripts than the two identified in the cDNA from peripheral blood lymphocyte strains. This difference might be due to the different sources of cells or the markedly lower expression levels of other transcripts, which prevented their detection through gel electrophoresis. The difference in the expression level of transcripts between patients and normal individuals indicates that variants at this splicing site may lead to changes in selective splicing behavior and disrupt the regulatory mechanism of selective splicing. The resulting changes in expression level may, in turn, cause OI. However, the alternative transcripts may indeed be insignificant in the process of collagen synthesis and may never mature to exist in the extracellular matrix. Studies have constructed plasmids and transfected single cell lines for 24 or 48 h to extract RNA for studying splice-site mutations (36–38). This method has been validated to be helpful for the pathogenicity assessment of splice-site mutations. Therefore, the present study also harvested RNA 48 h after plasmid transfection into a single cell line. However, due to the varying levels of gene expression in different cell lines, more cell lines are needed to confirm this finding. In addition, RNA extraction was performed only 48 h after plasmid transfection into 293T cells, so the expression of COL1A1 might not be comprehensive enough; the culture time after transfection could be extended to 72 h. We aim to continue to extract RNA from transfected cells 72 h later to observe the expression of this gene. Due to the inability to obtain tissues from the human body, we are currently unable to conduct western blotting experiments to assess the stability of the protein. More research is needed to detect changes in the expression of this protein.

OI not only seriously affects the quality of life of patients, but is also associated with an economic burden to families. In addition, no effective cure for this disorder currently exists. Prenatal diagnosis and in vitro fertilization combined with pre-implantation genetic testing can effectively prevent families with OI from having children with the same variant again. In-depth research on the genetic pathogenesis of OI will provide a theoretical basis for the prevention and treatment of this disorder, which has important clinical value and scientific significance.

Using WES technology, the present study identified a heterozygous variant, c.3531+1G>T, at the splicing site of intron 47 of the COL1A1 gene in a family with OI. The results revealed that this variant could lead to changes in splicing behavior and alter transcript expression levels. RT-PCR experiments on lymphocyte strains from the proband and an age-matched control showed that the c.3531+1G>T variant affected the splicing of intron 47. Minigene splicing assays also indicated that this variant site had an impact on intron 47 splicing. Thus, this variant was considered pathogenic for the proband. To the best of our knowledge, no functional study has yet been conducted regarding this variant and this variation has not been reported in the Chinese population. The present study is the first report of c.3531+1G>T in COL1A1 associated with OI in a Chinese family. It is also the first study regarding the effect of the c.3531+1G>T variant on the COL1A1 gene. Moreover, the OI phenotype caused by this variant is variable, ranging from mild (only one fracture and normal height) to moderate (15 fractures and short stature). These results may expand the spectrum of pathogenic variants associated with OI.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82101955), the Fujian Provincial Natural Science Foundation of China (grant no. 2023J05269), the General Fund Project of Xiamen Natural Science Foundation (grant no. 3502Z202373117), and the Fujian Provincial Health Technology Project (grant no. 2020GGB064).

Availability of data and materials

The data generated in the present study may be requested from the corresponding authors. The sequencing data generated in the present study may be found in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences under accession number GSA-Human: HRA011257 or at the following URL: https://ngdc.cncb.ac.cn/gsa–human/browse/HRA011257.

Authors' contributions

YH, YZ, LZ and YS designed the study, performed the laboratory experiments and bioinformatics analysis of WES. YZ, LZ, YS, XY and JX collected data and performed Sanger sequencing validation experiments. YH and YG confirm the authenticity of all the raw data. YH wrote the manuscript. LM and YG analyzed and interpreted the data, supervised the study and made revisions to the manuscript. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study fully complied with the tenets of The Declaration of Helsinki and was approved by the Ethics Board of the Women and Children's Hospital and the Experimental Animal Center of Xiamen University, China (approval no. KY-2022-090-K02). Written informed consent to participate in this study was provided by the participants, legal guardians or next of kin.

Patient consent for publication

The patients provide written informed consent, agreeing to publish their clinical manifestations and data.

Competing interests

The authors declare that they have no competing interests.

References