Download Phenotypic variability of osteogenesis imperfecta is not accounted

Phenotypic variability of osteogenesis imperfecta is not accounted for by disruption of splicing cis-elements
+1Kaneko, H; 1Kitoh, H, 1Matsuura, T; 1Masuda, A; 1Ito, M; 2Mottes, M; 3Rauch, F; 1Ishiguro, N; 1Ohno, K
+1Nagoya University Graduate School of Medicine, Nagoya, Japan, 2University of Verona, Verona, Italy,
3
Shriners Hospital for Children and McGill University, Montréal, Québec, Canada
[email protected]
INTRODUCTION:
Osteogenesis imperfecta (OI) is caused by mutations in COL1A1
on chromosome 17 or COL1A2 on chromosome 7. Similar mutations in
each gene exhibit widely variable phenotypes, and genotype-phenotype
correlations have not been fully elucidated.
Pre-mRNA is regulated by both intronic and exonic splicing ciselements. Both constitutively spliced and alternatively spliced exons
harbor exonic splicing enhancers (ESEs) and silencers (ESSs). A
mutation in the coding region is generally predicted to exert its
pathogenicity by compromising a functional amino acid, but nonsense,
missense, and even silent mutations potentially disrupt an ESE or ESS,
thereby resulting in aberrant splicing. More than 16% to 20% of exonic
mutations are predicted to disrupt an ESE.
Here, we hypothesized that preservation or disruption of an exonic
splicing cis-element determines the clinical phenotype. In a Japanese
family, hyperuricemia cosegregated with OI. In order to trace a
responsible gene for hyperuricemia, we looked for a founder haplotype
shared by the Japanese family and the previously reported Italian and
Canadian families with COL1A1 c.3235G>A.
METHODS:
Informed consent was obtained from all family members. The
current studies have been approved by the Institutional Review Boards
of the Nagoya University, the University of Verona, and the McGill
University. We performed mutation analysis of a Japanese family with
OI type I using genomic DNA and cDNA.
Five missense mutations (c.3226G>A, c.3226G>T, c.3235G>A,
c.3244G>T, and c.3253G>A) in exon 45 of COL1A1 cause mild to lethal
OI phenotypes. We predicted effects on pre-mRNA splicing of 18
sequence variants with or without each mutation in the presence or
absence of each of two SNPs (rs1800215 and rs1800217) in exon 45 of
COL1A1 using five web-based programs: ESEfinder 3.0, ESRsearch,
FAS-ESS, PESXs, and RESCUE-ESE. We constructed 18 variant
COL1A1 minigenes including exons 44-46 and the intervening introns
using the pcDNA3.1(+) mammalian expression vector (Fig. 2A). We
transfected each of minigene constructs into HEK293 cells. After 48
hours, we extracted total RNA and synthesized cDNA. We performed
RT-PCR using generic primers on pcDNA3.1.
To determine haplotypes of the Japanese, Italian, and Canadian
families with COL1A1 c.3235G>A, we genotyped three microsatellite
markers (D17S1293, D17S1319, and D17S788) and a SNP (rs2075554)
in intron 11 of COL1A1.
RESULTS:
A father and his three sons had blue sclera, dentinogenesis
imperfecta, and joint laxity (Fig. 1). Two sons (II-1 and II-3) had
histories of more than 10 fractures before age 13 years, but three other
members experienced no fracture. One son (II-1) had hearing loss from
age 10 years and hip joint deformities due to repeated femoral fractures.
We identified a dominant missense mutation, c.3235G>A in
COL1A1 exon 45, in four patients in the Japanese family (Fig. 1). We
asked if some mutations in COL1A1 exon 45 disrupt an exonic splicing
cis-element and result in phenotypic variability. Three programs showed
that five mutations and two SNP in exon 45 were predicted to affect 16
putative splicing cis-elements. We analyzed 18 minigenes, and found
that none affected pre-mRNA splicing (Fig. 2B).
In the Japanese family, hyperuricemia cosegregated with OI, but
not in the previously reported Italian and Canadian families with
c.3235G>A. We found that each family carried a unique haplotype (Fig.
1). Thus, hyperuricemia was possibly caused by a mutation in a gene on
the same chromosome as COL1A1. Two candidate genes are on the
same chr as COL1A1 at 17q21.31-q22. PRPSAP1 on chr 17q24-q25
encodes PAP39 and PRPSAP2 on chr 17p12-p11.2 encodes PAP41.
PAP39 and PAP41 are subunits of phosphoribosylpyrophosphate
synthetase that leads to urate production. Sequencing the entire coding
regions of both genes, however, revealed no mutation.
DISCUSSION:
Patients within the same family carrying the same mutation
sometimes exhibit variable OI phenotypes. In the Italian family with the
c.3235G>A mutation, the son suffered from many fractures, but his
mother did not have any. In the Canadian family with the same mutation,
the father was classified as OI type IV, whereas his daughter was OI
type I. Variable clinical phenotypes of the c.3235G>A mutation is
potentially due to differences in environmental factors or to SNPs in
disease-modifying genes.
Phenotypic variability of mutations in COL1A1 exon 45 is not
accounted for by splicing dysregulations. Our analysis suggests that the
currently available algorithms cannot efficiently predict ESEs.
To our knowledge, association of hyperuricemia with OI has been
reported in a single family, in which two of three OI patients had gouty
arthritis and hyperuricemia at young ages by Allen et al. (1955). The
cosegregated hyperuricemia is likely to have arisen from a mutation in a
yet unidentified gene on chromosome 17 in the Japanese family.
Figure 1. OI pedigrees and haplotypes of the Japanese, Italian and
Canadian families with the c.3235G>A mutation of COL1A1. The
clinical subtypes are all type I except for the Canadian father (V-1) who
exhibits type IV. The Japanese, Italian, and Canadian families carry their
unique haplotypes (shown by solid, dotted, and broken boxes,
respectively) that are not shared across different ethnic origins. The
D17S1293 genotype in the Canadian family is not informative.
Figure 2. Splicing assay using COL1A1 minigenes. (A) Schematic
representation of COL1A1 minigenes. (B) RT-PCR of minigenes
introduced into HEK293 cells. All constructs show a single fragment of
336 bp, indicating that COL1A1 exon 45 is not skipped in any constructs.
SNP1 and SNP2 are rs1800215 and rs1800217, respectively.
Untransfected cells are used as a negative control (NC).
Poster No. 559 • 56th Annual Meeting of the Orthopaedic Research Society