Osteogenesis Imperfecta Genotypes and Genotype-Phenotype Relationships - Osteogenesis Imperfecta - page 97

respectively. 47 Sequence variation which disrupts either
site normally leads to skipping of the exon adjacent to
the variant donor or acceptor site. This has a special sig-
nificance for the triple-helical region exons of the type
I collagen genes which are all precise multiples of 9 bp
and begin and end with complete codons for glycine
and Yaa-position amino acids, respectively. The introns
between these exons are said to be in “phase 0” and
exon skipping will always result in the maintenance of
the translational reading frame and the production of a
protein with an internal amino acid deletion.
The COL1A1 gene has 143 unique sequence variants
which interfere with splice sites at donor and acceptor
sites with almost equal frequency for the vast majority
of exons. The sequence variation subtypes are 124 sub-
stitutions, 14 deletions, 3 insertion / deletions, 1 insertion
and 1 duplication. Most of these occur in single indi-
viduals with the most recurrent being the substitutions
c.1155+1G>C and c.1299+1G>A which have each been
identified in five unrelated individuals. For COL1A2
there are 73 variants which affect splicing and which can
be subtyped as 59 substitutions, 12 deletions and 2 dupli-
cations. The most frequently recurring variants resulting
in OI are c.1197+5G>A, c.1557+3A>G and c.2835+1G>A
with four instances each.
Exon skipping might reasonably be predicted to lead
to a dominant-negative effect resulting in severe forms
of OI because triple helices would frequently contain one
or two α-chains with internal deletions. However, some-
what contrary to expectation, ~83% of exon-skipping
mutations in COL1A1 result in milder phenotypes of OI
I, OI I / IV or OI IV, and severe or lethal phenotypes of OI
III or OI II account for the remaining ~17%. By way of
contrast, COL1A2 harbors no splice site variants leading
to severe or lethal forms of OI. Apart from the several
instances affecting exon 6 and resulting in the arthrocha-
lasis form of EDS, the remaining exon-skipping muta-
tions yield predominantly OI I and OI IV phenotypes.
Interestingly, there are three published accounts of skip-
ping of exons 10 and 11 which result in phenotypes
which combine features of OI and EDS. 48-50
The large number of milder forms of OI resulting from
splice-site sequence variants can be explained in terms of
the creation of null alleles rather than the production of
truncated protein chains. Around 50% of sequence varia-
tion occurs at splice acceptor sites, predominantly in the
highly conserved “-1” or “-2” position of the splice-site
consensus sequence. It has been proposed that the tran-
sition G>A at the -1 position of an acceptor can result
in the effective shift of the splice site by one base if the
first base of the adjacent exon is a G, which is indeed the
case for all triple-helical domain exons. If the resulting
mutated splice site conforms sufficiently to the acceptor-
site consensus sequence, the result is to effectively
truncate the adjacent exon by a single base and create

TABLE 10.4 frameshifts in COL1A1 Causing severe

Phenotypes

DNA Variant Protein Variant OI Type

c.2268_2269del p.(Pro757Trpfs*23) III

c.3580_3581del p.(Ala1194Trpfs*25) III

c.3969dupT p.(Val1324Cysfs*105) II

c.4238_4248dup p.(Ser1417Metfs*14) II

c.4247delC p.(Thr1416Argfs*11) II

Frameshifts at five positions in COL1A1 yield OI types which are inconsistent with the
expected OI type I phenotype.

COL1A1 (133 unique variants in 177 patients), but much
less so in COL1A2 (six unique variants in six patients).
The COL1A1 variants are distributed across the majority
of exons and result predominantly in OI type I. Where
the OI phenotype is not type I, types IV or I / IV are most
common (13 patients), but types II (three patients) and
III (two patients) are also observed ( Table 10.4 ). All three
instances of type II OI 30,40,41 are explicable in terms of
the underlying mutations, which lie in exons 50 and 51,
creating PTCs through frameshifts in the final exon of
the gene which will not result in NMD. It is presumed
that the resulting truncated protein chains exert a severe
dominant-negative effect in all three instances. For the
two instances of type III OI 16,29 no such clear-cut explana-
tion is immediately obvious. Both cases result from two-
base deletions which might interfere with exonic splicing
enhancer (ESE) or exonic splicing silencer (ESS) motifs. 42
However, in silico analyses of the wild-type and variant
sequences using the ESEfinder43,44 43,44 and RegRNA 45 pro-
grams suggest no loss or gain of ESEs or ESSs because of
these deletions which might result in interference with
RNA splicing, leading perhaps to mis-splicing of RNA.
Ideally, any possible splicing effects resulting from these
deletions should be tested directly through the analysis
of RNAs from cultured cells.

SPLICE-SITE VARIANTS

Introns are removed from hnRNA by the process of
splicing to create functional mRNAs which comprise just
the exon sequences of the gene in question. Several dif-
ferent mRNAs can, in principle, be derived from a single
hnRNA primary transcript through the process of alter-
native splicing. However, the expression of COL1A1 and
COL1A2 does not entail any alternative splicing. The
different length mRNAs which are observed for these
genes 46 are the result of the use of multiple polyadenyl-
ation signals at which transcription terminates.
Correct splicing depends upon maintenance of highly
conserved sequence motifs at the 5′ and 3′ ends of each
intron, referred to as the “donor” and “acceptor” sites,