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skipping exon 6 of either gene, which encodes the region
that contains the amino-terminal propeptide cleavage
sequence. Except for mutations that result in skipping
exon 3 of each gene, which is not in the same frame as
most other exons, splice site mutations in the region that
encodes the amino-terminal propeptide have only been
identified in individuals if the outcome is haploinsuf-
ficiency. This reflects our incomplete knowledge of the
function of the propeptide. Of the 46 exons that encode
the triple-helical domain, splice site mutations that affect
the flanking regions, a total of 368 (184 × 2) sites would
result in OI if changed. The carboxyl-terminal propep-
tide encoding domain provides another region of more
than 800nt, both coding and flanking splicing domains,
that can be changed and result in all OI phenotypes.
Haploinsufficiency for COL1A1 is the most common
single general class of mutations that result in OI ( http: //
www.le.ac.uk / ge / collagen / ). With few exceptions these
mutations result in an OI type I phenotype. Type I pro-
collagen must have at least two pro-α1(I) chains, the
proteins encoded by COL1A1 . The molecule can accom-
modate three pro-α1(I) chains but not more than a single
proα2(I) chain. The reason for this requirement remains
unclear.
A third mechanism is mutations at splice sites. The
one predictable splice site alteration that leads to a pre-
mature termination codon is a -1G>A substitution at any
of the exons that encode the triple-helical domain. In this
region of the gene, each exon is a cassette that begins
with a G (part of a glycine codon). The splice acceptor
sequence can be generalized as (C / T / A)AG in which the
-3 position can be occupied by any nucleotide except G
but in which C and T are most frequent, and the -2 and
-1 positions are invariant as A and G, respectively. In
these introns, substitution of the -1 position G by A shifts
the splice acceptor site one nucleotide downstream by
incorporating the first G of the glycine codon into the
splice acceptor sequence. Although clearly a common
mechanism in collagens, which contain the triple-heli-
cal coding domains in cassettes, this is probably a gen-
eral mechanism as the majority of exons start with G.
Other substitutions at the -1 position are less predictable
in their outcome.
Splice donor site mutations are quite varied in their
outcome and single substitutions may give rise to mul-
tiple products. The outcome depends on whether the
exon is skipped, which usually results in an in-frame
deletion in type I collagen genes or if a cryptic donor site
is activated. This may depend, in part, on the order in
which introns are removed and the availability of usable
cryptic sites. At a statistical level, only one of three
potential sites is likely to result in an in-frame insertion
(use of a downstream intronic cryptic site) or deletion
(use of a cryptic site in the upstream exon). The major-
ity should result in insertion or deletion of a number of
nucleotides not divisible by 3 and so be out-of-frame.
The majority of donor site mutations result in creation
of a frameshift, consistent with the theoretical consider-
ations. Rarer mutation events can result in haploinsuf-
ficiency if they create splice acceptor sites (usually) that
are used in preference to the constitutive site. Exon dele-
tions are rare events in type I collagens because all but
two exons have a number of nucleotides divisible by 3.
Whole gene deletion appears to be a rare event but has
been noted in a few instances.
MECHA NISMS OF HAPLOINSUFFI CIENCY
Haploinsufficiency occurs through several mecha-
nisms, most of which lead to mRNA instability and loss
of the protein product from that allele. A second mecha-
nism results in an inability of proα chains to assemble in
the correct fashion. In this setting the chain is generally
very unstable. The effect is loss of the protein from that
allele and apparent haploinsufficiency. As noted later,
clinical outcomes of these various types of mutation
may differ.
Haploinsuffiency mutations in COL1A1 and COL1A2
lead to the introduction of premature termination
codons in the coding region of the mRNA. The first is
single nucleotide substitutions that change codons that
encode leucine (2), serine (2), tyrosine (2), cysteine (2),
tryptophan (1), arginine (2), glutamine (2), lysine, glu-
tamic acid (2) and glycine (1) to nonsense (termination)
codons. Substitutions in arginine codons seem to be
most frequent, in part because the substituted nucle-
otide, C, is part of a CG dinucleotide and subject to a
common mutational mechanism by which the C in a
CpG dinucleotide is modified. A second mechanism
is the insertion or deletion of nucleotides that are not
divisible by 3 (these would result in frame insertion or
deletion of amino acids). The most common location at
which such alterations occur is in runs of single nucleo-
tides, apparently as a result of replication slippage dur-
ing replication.
NONS ENSE MEDIATED MRNA D ECAY
Collagens conform to the general principle that
introduction of premature termination codons that are
separated by the constitutive termination codon by an
intron and 40-50 nucleotides result in nonsense medi-
ated mRNA instability. 3 This mechanism is activated
because in the process of normal splicing a set of pro-
teins, known as the junction complex, is deposited just
upstream from the former exon / intron boundary. If the
ribosome falls off the mRNA early in translation upon
reaching a premature termination codon, the proteins of
 
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