Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients
Zhytnik et al. Human Genomics
Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients
Lidiia Zhytnik 0
Katre Maasalu 0 2
Ene Reimann 1 3
Ele Prans 3
Sulev Kõks 1 3
Aare Märtson 0 2
0 Department of Traumatology and Orthopedics, University of Tartu , Puusepa 8, 51014 Tartu , Estonia
1 Centre of Translational Medicine, University of Tartu , Ravila 14a, 50411 Tartu , Estonia
2 Clinic of Traumatology and Orthopedics, Tartu University Hospital , Puusepa 8, 51014 Tartu , Estonia
3 Department of Pathophysiology, University of Tartu , Ravila 19, 50411 Tartu , Estonia
Background: Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country. Methods: We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5′UTR and 3′UTR regions, to identify causative OI mutations. Results: We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of proα1/2 chains. Conclusion: Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.
Osteogenesis Imperfecta; Collagen I; COL1A1; COL1A2; Sanger sequencing
Despite being a rare genetic bone fragility disorder,
osteogenesis imperfecta (OI) is among the most widely
occurring of rare congenital skeletal dysplasias [
prevalence is estimated 1/10,000–20,000 at birth [
is characterized by low bone mineral density, recurrent
fractures, skeletal deformations, and blue eye sclera [
]. Other remarkable features of OI include
Dentinogenesis Imperfecta, triangular face, hearing loss, joint laxity,
short stature, and easy bruising [
OI has many manifestations and is considered a group
of disorders. Phenotypes range from mild osteopenia to
severe deformities or even mortality. In 1979, Sillence
described four OI types (I–IV) according to phenotype
]. Recent updated classification distinguishes
three additional types with specific histologies (V–VII)
]. Genetic OI classification considers every OI gene
as a separate OI type and so far includes OI types
4, 8, 9
The genetics of the disorder reflect the complexity of
the OI phenotype range. Up to 21 different genes have
been associated with occurrence of OI [
studies have shown that the primary cause of OI are
mutations in the COL1A1/2 genes, which code
procollagen type I α1 and α2 chains, respectively . Despite
the approximately 1500 mutations already described in
collagen type I genes, investigators continue to report
novel mutations [
]. Moreover, there is still some
controversy regarding the proportion of collagen
mutations reported in different populations, which have
ranged from 60 to 95% [
]. In this context, we
believe that population-based studies of OI genetics
might broaden current knowledge of collagen I
mutations and OI.
Due to Estonia’s small population (1.3 million) and
centered treatment, follow-up, and research of all OI
patients at the OI Center of the Traumatology and
Orthopedics Clinic, Tartu University (TU) Hospital, it was
possible to perform analysis of COL1A1/2 mutations
among the whole Estonian OI population [
we describe for the first time the mutational spectrum of
COL1A1/2 genes among 30 unrelated OI patients, from
30 Estonian OI families, which we estimate to constitute
~ 100% of OI cases in Estonia.
The patients included in the study are treated and
followed-up by the OI Center of the Traumatology and
Orthopedics Clinic, TU Hospital.
A total of 30 OI patients from 30 unrelated families
were included in the study. Data regarding the OI types
of the subjects were obtained from the medical records
of TU Hospital [
]. All new OI cases across Estonia are
registered by and treated at TU Hospital’s OI Center.
Thus, it can be estimated that as of May 2017, the
current patient cohort represented ~ 100% of the
Estonian OI population.
No patient came from a consanguineous family.
Mutational analysis of the COL1A1/2 genes was performed
on a younger affected member of every OI family
included in the study.
In accordance with the Declaration of Helsinki, all
patients or their legal representatives signed an informed
consent form prior to participation. The study was
approved by the University of Tartu’s Ethical Review
Committee on Human Research (permit no. 221/M-34).
Genealogical data of OI history in the family,
consanguinity, and miscarriages was obtained from each
patient or their representative. We constructed pedigree
trees per kindred using the “Kinship2” package in R
Mutational analysis of the COL1A1/2 genes
Genomic DNA (gDNA) was purified from 3 ml of
ethylenediaminetetraacetic acid (EDTA) preserved whole
blood samples—stored at −80 °C—using a Gentra
Puregene Blood Kit (Quiagen, Germany) following the
PCR amplification and Sanger sequencing were
performed as described previously [
]. Sequence products
were analyzed using Applied Biosystems’ Sequence
Scanner v1.0 and Mutation Surveyor DNA Variant analysis
software v5.0.1. (Softgenetics, USA) and aligned to the
GenBank human reference genome sequences of
COL1A1 (gDNA NG_007400.1, complementary (cDNA)
NM_000088.3), and COL1A2 (gDNA NG_007405.1,
cDNA NM_000089.3). Raw sequencing data are available
from the authors upon request. We focused on
nonsynonymous and splice-site variants absent from the
publicly available normal datasets (including dbSNP135
and the 1000 Genomes Project) [
]. We used the
PolyPhen-2, SIFT, and MutationTaster software tools to
predict the functional effects and pathogenicity of
]. Variants absent from the osteogenesis
imperfecta mutation database were considered novel
All statistical analyses were carried out with R v3.3.2.
software (R Team, Austria) [
]. To assess the
distribution of COL1A1/2 mutations and compare them to other
studied OI populations, percentage differences were
Mutational analysis of the COL1A1/2 genes of Estonian
OI patients highlighted OI causative mutations in 26 of
30 patients (86.67%) (Fig. 1a). The number of patients
harboring COL1A1 mutations was 20 (76.92%); COL1A2
mutations were found in 6 patients (23.08%) (Fig. 1b). A
list of the mutations and their characteristics can be
found in Table 1.
The number of novel mutations was 13/26 (50%) (Table
1). Half of the COL1A1 and COL1A2 mutations appeared
to be undescribed in the collagen type I mutation
database. Patient EE26 had a heterozygous non-synonymous
rs1800215 SNP (p.Ala1075Thr) in the COL1A1 gene,
which was described before as a benign variant (data not
Twenty-five mutations had an autosomal dominant
inheritance pattern (Table 1). Of these, eight patients had
no previous history of OI in the family. Thus, we
assumed that their parents and relatives, who did not
have any clinical features of OI, are not carriers of these
Patient EE07 had a recessive missense mutation.
Mutational analysis showed that their parents are not
carriers of the mutation, which confirmed the de novo
nature of the mutation.
We found 12/26 mutations (46.15%) had altering
splice sites, 10 and 2 in the COL1A1 and COL1A2 genes,
respectively. One of the patients harbored a deletion
capturing both coding and intronic sequence, in
exonintron 34 (EE30). Nonsense mutations were present in 6
patients (23.08%), all in the COL1A1 gene. Overall,
quantitative mutations were present in 18 patients (16 in
COL1A1 and 2 in COL1A2 genes) (Fig. 2).
Missense mutations, associated with collagen I quality
defects, were indicated in eight patients (30.77%), four in
COL1A1 and four in COL1A2 genes. Of these, seven
were Glycine substitutions (four of the COL1A2 and
three of the COL1A1 missense mutations). In four cases,
Glycine was substituted with Serine, two in the COL1A1
and COL1A2 genes, respectively (Fig. 2).
A c.3262G>T (COL1A1) mutation was detected in two
patients (EE21 and EE22), who were thought to be
unrelated (Table 1). Investigation of the pedigree trees
revealed a distant relationship between the families four
generations back, of which the patients were not aware.
Two identical splice site mutations at c.1821 + 1G>A
(COL1A1) in intron 26 were identified in patients EE02
(type III OI) and EE05 (type I OI) (Table 1). This
mutation arose independently in the patients and caused
phenotypes of different severity.
Collagen I mutations were found in 26/30 (87%) studied
OI patients. Previous findings have suggested collagen
mutations ranging from 60 to 90% among different OI
populations and study cohorts [
]. In a Finnish OI
study, 90.7% of patients harbored collagen I mutations
], which is higher than we found among Estonian OI
patients. In Pollitt et al.’s study, collagen I mutations
were revealed in 75% of OI patients [
], which is
slightly lower than our Estonian cohort. Our data is in
good agreement with research on the genetic
epidemiology of the Swedish OI population, of which 87% had
collagen I mutations [
]. The results of our study are
also in concordance with Bardai et al.’s recent study of a
large number (598) of OI individuals, where collagen
type I mutations were found in 86% of OI patients of all
types and different ethnic groups [
In some population studies, the amount of collagen I
mutations were also lower. For example, in 51.4% of
Taiwanese patients (N = 72), 52.2% of Korean patients
(N = 67), and 59.4% of Vietnamese OI patients (N = 91)
25, 37, 38
]. Due to the difficulties in arranging large
cross-population studies of a rare disorder in populous
countries, results can often be fragmented, which
complicates population-wide estimates [
questions about the lower collagen type I mutational
pattern of OI patients from Asian populations remain.
The proportions of COL1A1 and COL1A2 in Estonian,
Finnish, and Swedish OI populations were surprisingly
similar, 77 and 23%, 78 and 22%, and 79 and 21%,
]. Similar values were reported by
Pollitt et al., where 77% of mutations occurred in the
COL1A1 and 23% in the COL1A2 gene (N = 83) [
Patients with de novo mutations and without OI history in the family are marked with an octothorp (#). Novel mutations unreported in the collagen type I variant
database (http://www.le.ac.uk/ge/collagen/) are marked with an asterisk (*). In cases of heterozygous mutation, both the wild type and the mutated allele are
indicated after an arrow (>)
Bardai et al.’s 2016 study, 69% were COL1A1 and 31%
COL1A2 mutations, which is similar to the
beforementioned results [
The Estonian cohort also has a high proportion of
quantitative mutations compared to qualitative collagen
mutations, 69 and 31%, respectively. In the Finnish OI
cohort, 67% of mutations were quantitative and 33%
]. In the work of Pollitt et al., 35% of
mutations were qualitative and 65% quantitative [
the Swedish population, the proportions were almost
equal (53 and 47%) [
]. Interestingly, we found only
two quantitative mutations in the COL1A2 gene, which
matches previous reports about comparatively lower
numbers of quantitative mutations of this gene [
Due to the higher number of mutations leading to
haploinsufficiency in the COL1A1 gene compared to the
COL1A2 gene, patients harboring mutations in the
COL1A1 gene had milder phenotypes (I, IV) compared
to patients with COL1A2 mutations (type III, except
EE20 who had a splice site mutation and OI type I).
Glycine substitutions composed the vast majority of
missense mutations (7 of 8 cases), with serine being the
most substituted amino acid (4 of 7 cases), which
supports previous findings. Curiously, all missense
mutations were situated in triple helical chain domains
(aa residues 162–1218 α1; aa residues 80–1102 α2) of
COL1A1/2 gene products. Only one mutation (patient
EE07 with OI type II) altered the “lethal cluster”
proposed by Marini et al. [
Half of the mutations (50%) we found appeared to be
novel. Despite the numerous works on collagen I
mutations and a growing list of identified mutations, the
number of revealed novel variants was high, which underlines
the individual nature of OI mutations [
19, 35, 36
]. Half of
the glycine substitutions (4 of 7) were even absent from
the collagen I mutational database.
Sillence OI type
Despite sharing of the same mutation, patients may
develop different phenotypes, as in the case of patients
EE02 and EE05, who had type III and I OI, respectively.
Genotype-phenotype correlations remain an unresolved
issue in our understanding of OI. Cases of inter- and
intra-familial OI diversity are not rare. Not only genetics,
but additional factors, such as epigenetics and
environment might contribute to the development of specific
OI phenotypes. This leads to many questions and the
need to further investigate potential OI factors.
Sanger sequencing is a powerful and accurate method
of mutational analysis and allows the identification of
frameshift, and missense and nonsense mutations in the
coding regions of genes. Moreover, due to the special
design of the primers distant from intron-exon junction
regions, we could asses splice site mutations of the
COL1A1/2 genes, which are the cause of quantitative
collagen defects. However, the current study had some
limitations. We could not identify whole gene or exon
deletions and duplications, which could have slightly
reduced the number of discovered COL1A1/2 mutations.
In addition, due to the small population size of Estonia,
our cohort was limited. We cannot exclude the
possibility that the small sample size might be the cause of
differences compared to the results of other studies.
This paper has described the mutational spectrum of
COL1A1/2 genes among 30 Estonian OI patients, which
were estimated to represent ~ 100% of OI families in
Estonia at the time. We identified collagen I mutations
in 87% of Estonian OI families. The number of
quantitative mutations (69%) was high compared to
other European OI cohorts. All missense mutations of
our Estonian patients altered the triple helical chain
domain of α1 and α2 procollagen chains. One mutation
was situated in the lethal cluster. A normal distribution
of novel collagen mutations (50%) among the COL1A1
(77%) and COL1A2 (23%) genes, and mostly glycine
substitutions were observed, compared to other OI cohorts
of Northern Europe. Four patients that showed no
collagen type I mutations will be further studied using whole
exome sequencing analysis to identify disease causing
3’UTR: Three prime untranslated region; 5’UTR: Five prime untranslated
region; cDNA: Complementary DNA; EDTA: Ethylenediaminetetraacetic acid;
gDNA: Genomic DNA; OI: Osteogenesis Imperfecta; TU: University of Tartu
We would like to thank the following people and organizations for their help
and support with data collection: workers of the Department of Traumatology
and Orthopedics and Department of Pathophysiology, University of Tartu, and
Ardo Birk and Madis Karu for the development of the online OI database of the
Clinic of Traumatology and Orthopedics, TU Hospital.
This study was supported by the Estonian Science Agency project IUT20-46
(TARBS14046I), the European Regional Development Fund and the Archimedes
Foundation to the Centre of Excellence on Translational Medicine, the University
of Tartu’s Development Fund, University of Tartu’s Baseline Funding, and the
HypOrth Project funded by the European Union’s 7th Framework Programme
grant agreement no. 602398.
Availability of data and materials
The datasets used and analyzed during the current study are available from
the corresponding author upon reasonable request.
LZ conceived the study, carried out the genetic studies, performed the data
analysis, participated in the design of the study, and drafted the manuscript. EP,
SK, and ER carried out the genetic studies, performed the data analysis, and
helped to draft the manuscript. KM participated in the design of the study,
interacted with the patients, coordinated the blood sample collection, and
helped to draft the manuscript and perform analysis. SK and AM participated in
the designing of the study, coordinated the data interpretation and statistical
analysis, and helped to draft the manuscript. All authors read and approved the
Ethics approval and consent to participate
The current study was conducted in accordance with the Helsinki
Declaration and received approval from 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 were
obtained prior to inclusion to the study.
Consent for publication
The authors declare that they have no competing interests.
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