Mutation analysis of the COL1A1 and COL1A2 genes in Vietnamese patients with osteogenesis imperfecta
Ho Duy et al. Human Genomics
Mutation analysis of the COL1A1 and COL1A2 genes in Vietnamese patients with osteogenesis imperfecta
Binh Ho Duy 0 1 2
Lidiia Zhytnik 0
Katre Maasalu 0 2
Ivo Kändla 3
Ele Prans 3
Ene Reimann 3
Aare Märtson 0 2
Sulev Kõks 3 4
0 Department of Traumatology and Orthopaedics, University of Tartu , Puusepa 8, 51014 Tartu , Estonia
1 Hue University of Medicine and Pharmacy, Hue University , 06 Ngo Quyen, Hue city 530000 , Vietnam
2 Clinic of Traumatology and Orthopaedics, Tartu University Hospital , Puusepa 8, 51014 Tartu , Estonia
3 Department of Pathophysiology, University of Tartu , Ravila 19, Tartu 50411 , Estonia
4 Centre of Translational Medicine, University of Tartu , Ravila 14a, Tartu 50411 , Estonia
Background: The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI. Methods: Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish's osteogenesis imperfecta mutation database. Results: The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients. Conclusion: Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.
Osteogenesis imperfecta; Collagen type I; Bone fragility; Sanger sequencing
Osteogenesis imperfecta (OI) is associated with high
genetic heterogeneity. To date, mutations in 16 different
genes have been found to cause OI phenotypes of
varying severity [
]. About 90 % of the mutations are related
to alterations in the COL1A1 and COL1A2 genes,
located at chromosome 17q21.33 and 7q21.3, respectively
]. These genes code for the α1/α2 chains of type 1
]. It was hypothesized that due to the
presence of two α1 and one α2 chains in the procollagen
triple helix, the COL1A1 is more susceptible to
mutation, as more α1 chains are implemented in the collagen
fibrils. COL1A1 gene mutations are more pathogenic
and cause OI more often than COL1A2 gene mutations.
One third of glycine (Gly) substitutions in the COL1A1
gene are lethal, whereas only 1/5 of Gly pathogenic
variants in the COL1A2 gene are fatal [
]. The collagen
primary structure differs with an obligatory presence of Gly
residues, the smallest amino acid, in every third position
of an α chain, composing (Gly-X-Y)n repetitions, where
X and Y are random amino acids [
]. The substitution of
Gly positioned in the center of the triple helix by a
different amino acid would prevent interchain hydrogen
bond formation between the NH-group of Gly and the
CO-group in the X-position of a neighboring chain.
Moreover, substitution of Gly residues with branched
nonpolar or charged amino acids changes the helix to
bulky and unstructured [
]. In this way, helix strength
and stability decrease, which are crucially important for
protein function [
Type 1 collagen is one of the most abundant proteins
in the human body. It is a structural component of the
bone, skin, tendons, cornea, and blood vessel walls and
other connective tissues [
]. OI is generally caused by
qualitative or quantitative collagen type I defects [
More than 2500 OI mutations have been found in type I
collagen genes, which can cause a wide range of OI
phenotypes that range in severity from mild to severe
] (http://www.le.ac.uk/ge/collagen/). Previous
studies have shown that COL1A1/2 mutations account for up
to 85–90 % of all OI causative mutations, whereas only
10–15 % of OI mutations occur in non-collagenous genes
2, 11, 12
]. While in more recent studies, many new
genetic causes have been described, the mutations in the
COL1A1/2 genes remain a common origin of OI [
However, there is a lack of systematic information
regarding the mutational characteristics of OI patients. In
addition, the genetics of Vietnamese OI patients has not
been studied before. Our main aim with the current study
was to perform mutational analysis of the COL1A1 and
COL1A2 genes among unrelated OI patients of Vietnamese
origin. We applied a systematic approach to characterizing
the mutation profiles of these two genes.
Materials and methods
The study was conducted in accordance with the Helsinki
Declaration and received approval from the ethical review
board of Hue University Hospital (approval no.
75/CNBVYD) and the Ethical Review Committee on Human
Research of the University of Tartu (permit no. 221/M-34).
Patients were selected from the Vietnamese database of
osteogenesis imperfecta patients. The database includes
information on 146 OI patients from 120 OI families and also
about their healthy family members. A total of 91 unrelated
OI patients were included in the study. Informed written
consent from the patients or their legal representatives was
obtained prior to inclusion to the study. Investigators then
contacted patients in order to conduct an interview,
perform a clinical examination, and collect blood samples,
including blood samples from parents, siblings, and close
relatives. Genomic DNA was extracted from
EDTApreserved blood according to standard high-salt extraction
methods, stored at −80 °C, and analyzed at the University
of Tartu, Estonia.
DNA samples were amplified using a polymerase chain
reaction (PCR) with 25 specially designed primer pairs
covering the 5′ UTR and 3′ UTR regions and 51 exons
of the COL1A1 gene; 36 primer pairs covering the 5′
UTR and 3′ UTR regions and 52 exons of the COL1A2
gene. The PCR reaction was performed in a total volume
of 20 μl, which included 4 μl of 5× HOT FIREPol® Blend
Master Mix Ready to Load with 7.5 mM MgCl2 (Solis
BioDyne, Estonia), 1 μl each of forward and reverse
primer (5 pmol), and 1 μl of gDNA (50 ng). PCR reaction
was performed with a Thermal Cycler (Applied
Biosystems, USA) PCR machine. The PCR touchdown program
was used as follows for the reaction of amplification:
1 = 95.0°; 15:00 min
2 = 95.0°; 0:25 min
3 = 64.0°; 0:30 min
4 = 72.0°; 0:40 min
5 = go to 2.4 times
6 = 95.0°; 0:25 min
7 = 62.0°; 0:30 min
8 = 72.0°; 0:40 min
9 = go to 6.30 times
10 = 72.0°; 5:00 min
11 = 6.0°; forever
Amplified PCR products were electrophoresed through a
1.5 % agarose gel, to control the quality of fragments. The
PCR products then purified with exonuclease I and shrimp
alkaline phosphatase (Thermo Fisher Scientific, USA).
Sanger sequencing reactions were performed on the
purified PCR fragments using a BigDye® Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, USA). Reactions were
processed on the ABI3730xl instrument.
Sequence reads were analyzed using Applied
Biosystems’ Sequence Scanner v1.0 and aligned to the human
reference genome Local Reference Genomic sequence
LGR_1 and GR_2. Raw sequencing data are available from
authors upon request. Sequence analysis and pathogenic
variant identification were performed with Mutation
Surveyor DNA variant analysis software (Softgenetics, USA).
Prediction of mutation’s pathogenicity was performed
using Alamut Visual software (Interactive Biosoftware,
France). Variants were checked against the osteogenesis
imperfecta mutation database (http://www.le.ac.uk/ge/col
lagen/). The pathogenicity of the pathogenic variants was
predicted with SIFT score [
We studied 42 female and 49 male OI patients. To
characterize the OI patients’ clinical features, all
participants underwent clinical and physical examinations, and
their medical records were reviewed. Cases were described
according to the Sillence classification (types I–IV) [
Fifty-four patients were found to have COL1A1/2
mutations, 33 with COL1A1 and 21 with COL1A2; this
equated to 36.3 and 23.1 % of patients, respectively, totaling
59.4 % of the studied OI cases exhibiting collagen type I
mutations. Thirty-four pathogenic variants in the COL1A1
gene (missense = 23, nonsense = 4, splice site = 7) and 22
pathogenic variants in the COL1A2 gene (missense = 21,
splice site = 1) were identified (patients VN01 and VN47
were carriers of double pathogenic variants in both the
COL1A1/2 genes) (Fig. 1; Tables 1 and 2). According to
Dalgliesh database, 17 COL1A1 and 10 COL1A2 variants
have not been reported before (Tables 1 and 2). De novo
mutations were observed in 50 % (17/34) of COL1A1
variants and 45.5 % (10/22) of COL1A2 variants. All
mutations were highly pathogenic, with a SIFT score of 0.0 and
rarely 0.1, and located in regions of high conservation.
In our study, we performed mutational analysis of 91
Vietnamese patients clinically diagnosed with OI (types
I–IV). Thirty-three patients had 34 pathogenic variants
of the COL1A1 gene, and 21 patients had 22 pathogenic
variants of the COL1A2 gene, equating to a total of 54/
91 (59.4 %) patients with COL1A1/2 pathogenic variants.
Previous studies have indicated that nearly 90 % of all
OI mutations appear in the COL1A1 and COL1A2 genes
]. However, reported collagen type I mutational
rates vary between different populations from 58 to
96 % [
We identified the substitution of Gly residuals in 17
out of 23 missense COL1A1 mutations and 19 of 21
missense COL1A2 pathogenic variants. Gly substitutions
composed 36/56 (64.3 %) of COL1A1/2 pathogenic
variants. It has been hypothesized that the majority of the
clinically severe forms of OI are caused by Gly missense
]. However, there may exist a complex
relationship between OI pathogenic variant and OI
severity, whereby genetic, epigenetic, and environmental
factors altogether affect the phenotype [
Our research showed that out of 36 glycine
substitutions, serine was the most prevalent (23/36; 63.9 %),
followed by valine (4/36; 11.1 %), and cysteine and aspartic
acid (3/36 cases each). Previous studies have suggested
that glycine substitutions by cysteine often cause a greater
severity of OI phenotype, and glycine substitutions by
arginine were often fatal [
]. However, there are alternative
reports that also suggest serine is the most common
substitutional residue of Gly (72 % among Chinese OI
]. Aspartic acid substituted Gly in 40 % of
Taiwanese OI patients [
]. The cause of variation in
amino acid substitutions among populations of different
geographical regions is still unclear.
In our research, intronic variants were represented by
seven splice site mutations; other research has reported
intronic variants among 7/56 of Chinese OI patients
]. These mutations may cause exon skipping, intronic
inclusion, and activation of cryptic sites [
]. In addition,
analyses identified two nonsense mutations located in
exons 52 and 37. Nonsense and splice site mutations are
associated with haploinsufficiency, and as a result,
quantitative collagen type I defects and a mild–moderate OI
phenotype (type I/IV).
Patients VN01, VN34, VN40, and VN49 had the same
heterozygous mutation: c.2461G > A (p.Gly821Ser) in
Total number of
Splice site (7)
Splice site (1)
Gly residue (17)
Non-Gly residue (6)
exon 37 of the COL1A1 gene. With respect to clinical
severity, these patients showed nearly the same
manifestations (clinical types I and IV). However, previous
studies have described OI patients with different
clinical features, despite their being carriers of the
c.2461G > A mutation. Current data highlights the
complexity of OI genotype–phenotype correlations. It
is not yet possible to predict disorder severity based
only on mutational analysis data.
Families VN88 and VN89 shared the same
heterozygous COL1A1 c.2005G > A (p.Ala669Thr) pathogenic
variant in exon 30. Two patients had the same
pathogenic variant and level of OI severity (type IV).
Similar cases of variant reoccurance have been
described before by Zhang et al. and Lee et al. in both
COL1A1/2 genes [
]. However, OI pathogenic
variants are usually unique and rarely repeated among
different families .
c.2261G > GT*
c.1072G > GT
c.1630G > GA*
c.1090G > GA
c.3034G > GA
c.2569C > CA
c.1451G > GA
c.1729G > GA*
c.1009G > GA
c.1378G > GA
c.1964G > GT*
c.1981G > GC*
c.874G > GA
c.982G > GA
c.2503G > GA
c.792 + 1G > GA
c.2791G > GA*
c.838G > GT*
c.2791G > GA*
c.892G > GT*
c.2538G > GT*
Mutations unreported in the Dalgliesh’s OI database are marked with an asterisk (*). In the case of heterozygous mutation, both the wild type and mutated alleles
are indicated after an arrow (>)
Genetic analysis revealed the presence of two
heterozygous COL1A1 mutations: exon 37 c.2461G > A
(p.Gly821Ser) and exon 30 c.2005G > A (p.Ala669Thr)
in patient VN01. Both pathogenic variants were shared
by other unrelated patients among our study cohort.
Patient VN47 had two heterozygous COL1A2
pathogenic variants: exon 46 c.3034G > GA (p.Gly1012Ser)
and exon 41 c.2569C > CA (p.Pro857Thr). Takagi et al.
reported one case of severe OI (types II–III) due to a
double substitution of glycine residues in the COL1A2
gene (p.Gly208Glu and p.Gly235Asp), located on the
same allele [
]. Our patients had only one substituted
Gly residue in the COL1A1 gene and a mild phenotype
(VN01) and moderate phenotype (VN47) based on the
Of the 56 mutations found during our research, 17
COL1A1 and 10 COL1A2 variants (27/56 pathogenic
variants; 48.2 %) were not present in Dalgleish’s OI
mutation database (Tables 1 and 2) [https://oi.gene.le.a
c.uk/home.php?select_db=COL1A2]. The percentage of
new variants among our patients was higher than in
previous studies [
16, 22, 25, 26
]. The novelty of the
pathogenic variants highlights the originality of the
genetic epidemiology of the Vietnamese OI population.
Half of Vietnamese OI patients are carriers of rare
recessive non-collagenous OI pathogenic variants, which will
be further identified with the whole exome sequencing
analysis and reported in a future paper.
According to our data, more OI causative
pathogenic variants occurred in the COL1A1 gene than the
COL1A2 gene. Mutation hotspots were observed in
intron 1; exons 8, 14–15, 17–20, 30, 33, 34, 37, and
52 of the COL1A1 gene; and exons 17–49 of the
COL1A2 gene (Fig. 2). Products of the COL1A1/2
gene consisted of signal peptide, N-terminal
propeptide, collagen alpha I/II chain triple helical domain,
and C-terminal propeptide (COLFI). COLFI controls
procollagen intracellular assembly and the
extracellular assembly of collagen fibrils. Mutation hotspots
were situated in the regions that tolerate amino acid
substitutions, and pathogenic variant resulted in an
altered protein, but the organisms were still able to
survive. Gaps in the mutation map connected to
regions with crucial functions can however lead to fatal
Sequencing primers for the performed Sanger
sequencing of the COL1A1 and COL1A2 genes in patients with
clinical signs of osteogenesis imperfecta were designed
far from intron-exon splice sites, which allowed the
identifying of splice site, missense, frameshift, and
nonsense mutations in the exons of the COL1A1/2 genes.
The gold standard of sequencing, the Sanger method,
has an accuracy of approximately 99.9 % [
it has limitations in identifying whole genes and exon
duplications and deletions. Therefore, the number of
COL1A1/2 pathogenic variants in the studied OI
patients might have been underestimated.
We must also take into consideration that the
percentage of collagen pathogenic variants among osteogenesis
imperfecta patients may vary between studies due to
their different sample sizes. However, we cannot exclude
the possibility that the Vietnamese population has lower
rates of collagenous OI pathogenic variants, and a
unique OI mutational profile with higher levels of rare
non-collagenous pathogenic variants, compared to other
In the current study, we conducted mutational analysis of
the COL1A1 and COL1A2 genes among 91 Vietnamese
patients with osteogenesis imperfecta. After sequencing of
the COL1A1 and COL1A2 genes, we found 56 mutations
in 54 patients (59.4 % of patients). Our data showed a
lower number of collagen OI pathogenic variants in these
Vietnamese patients compared to reported rates for other
Asian OI populations. The OI mutational profile of the
Vietnamese population is likely unique and is related to
the presence of a high number of recessive mutations in
non-collagenous OI genes. Further analysis of patients
negative for collagen OI mutations is needed in order to
reveal unidentified OI genotypes from the sample.
3′ UTR, 3′ untranslated region; 5′ UTR, 5′ untranslated region; COLFI, fibrillary
collagen C-terminal domain; EDTA, ethylenediaminetetraacetic acid; gDNA,
genomic DNA; OI, osteogenesis imperfecta; PCR, polymerase chain reaction
We would like to thank the following people and organizations for their help
and support with data collection: The Vietnamese National Hospital of
Pediatrics; Hanoi OI Center; OI Booming Diamond Center in Ho Chi Minh City;
Hue University Hospital; and The University of Tartu. This research would not
have been possible without the support, teaching, and cooperation of the
Department of Traumatology and Orthopaedics and Department of
Pathophysiology of University of Tartu.
This work was supported by institutional research funding IUT20-46 of the
Estonian Ministry of Education and Research and the European Union’s European
Regional Development Fund Programme “Supporting international cooperation
in R&D” projects “EVMED” and “DIOXMED.” The research leading to these results
has received funding from the European Union’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 602398.
Availability of data and materials
The dataset supporting the conclusions of this article, including raw
sequencing and clinical data, is available from authors upon request.
HDB conceived the study, participated in its design, interacted with the
patients, coordinated the blood sample collection, and drafted the
manuscript. LZ, IK, EP, SK, ER carried out the genetic studies, performed the
data analysis, and helped to draft the manuscript. KM participated in its
design, interacted with the patients, coordinated the blood sample
collection, and helped to draft the manuscript. SK and AM participated in
the design of the study, coordinated the data interpretation and statistical
analysis and helped to draft the manuscript. All authors read and approved
the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was conducted in accordance with the Helsinki Declaration and
received approval from the ethical review board of Hue University Hospital
(approval no. 75/CN-BVYD) and the Ethical Review Committee on Human
Research of the University of Tartu (permit no. 221/M-34). Informed written
consent from the patients or their legal representatives was obtained prior
to inclusion to the study.
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