4. Discussion
Different COL7A1 gene mutations result in dystrophic
epidermolysis bullosa disorder. DEB patients are characterized by
affected dermis-epidermis conjunction and they are extremely prone to
blistering by mechanical stimulation. The affected genome location of
DEB patients was localized on the short arm of chromosome 1 by genetic
linkage analysis. This region encompasses COL7A1 gene which has
118 exons and span 32 Kb of human genome (9, 10). collagenVII is a
homotrimer molecule consists of three identical α1 chains with various
Gly-X-Y repetition. The glycine, with the single hydrogen atom in the
side chain, is the smallest amino acid which provides the flexibility in
collagen molecule by allowing flexible bending of the polypeptide. The
Glycine substitution (GS) mutation could give rise to both autosomal
recessive and dominant form of DEB (4).
Here, we report two families with GS mutations in different region ofCOL7A1 gene which were inherited in autosomal recessive mode in
family I (COL7A1 , NM_000094, c.5018G>A, p.G1673E)
and autosomal dominant in family V (COL7A1 , NM_000094:
c.6101G>A, G2034E). Glycine has critical structural roles
in the triple helical structure. The substitution of glycine for large
amino acids might destabilize triple helix structure of collagen
molecule. The recessive GS mutation inhibits α-chain synthesis
completely. According to protein details of UniProt database amino acids
1254-2784 constitute the triple helix region of the protein. The glycine
1673 and 2034 is located in this region, hence affect the triple helical
structure. Glycine substitutions in collagen VII have adverse
consequences on the formation and function of anchoring fibrils.
Bruckner-Tuderman et al identified G2034R mutation interfering
with protein folding and result in collagen VII accumulation (11).
According to the mutation taster prediction, c.5018G>A
mutation exerts its effect on splicing site by producing cryptic donor
splicing site in exon 54 which affect downstream structures such as
disulfide bond and NC2 region of the protein. In 2011, G1673R was
reported as disease-causing substitution in DEB patients (12).
Therefore, this mutation cause RDEB and is disease causing variant.
Another GS mutation was observed in family V. The p.G2034E substitution
was reported as a disease-causing mutation in dominant mode (13). Here,
the results also revealed heterozygous mutation in patient. However, nor
mother neither father had this mutant allele. Hence, this mutation was
inherited as autosomal dominant manner and it seemed that G2034E was a
de novo event due to no family history of EB. In addition, this mutation
can affect downstream structure by producing cryptic donor splice site
mutation.
Mild DEB patient without any positive family history usually were known
as de novo or sporadic DDEB cases. De novo individuals often had
dominant mutation in COL7A1 and transfer the mutation to
offspring with 50% probability. Various missense mutations of this
region or flanking segments are reported in DEB patients which showed
the importance of this region of type VII collagen molecule (4). De novo
GS leading to DDEB has been reported in the following positions:
G1775D (14) , G2012S (15), G2012D (16), G2028R (17), G2034R ,G2034W(18),
G2040V (19), G2067R (14), G2076D (20), G2079E (21),G2348R (22).
Substitution of glycine in 2034 position for arginine and tryptophan
amino acids were previously reported in affected individuals (18) and in
this study G2034E was identified in our patient.
The deleterious mutation of family II (COL7A1 , NM_000094,
c.425A>G, p.K142R) was previously reported as the first
mutation which leads to splicing deficiency of COL7A1 . It is
located in the vicinity of the donor splice site (position -2 of the
intron 3) which produce three aberrant mRNA depending on the skipping of
exon 3, activation of cryptic donor splice site within exon 3 or
retention of entire intron 3. Gardella, et al identified a
premature termination codon (PTC) before the end of exon 4 in all
altered transcripts resulting in truncated pro- α1 (VII) chains. This
chains are likely to degrade by nonsense mediated decay (NMD) mechanism
and do not include in COLVII anchoring fibrils (23). Nonsense-mediated
decay (NMD) is the mRNA surveillance mechanism which degrades mRNA with
in-frame PTC to protect cells from dominant negative effect (24).
Haplotype analysis of COL7A1 gene have been identified some
recurrent mutations in distinct patients with similar allelic
background. c.425A>G mutation was found in Italy and
central European cases as recurrent one (4).Lysine 142 is a conserved
amino acid during evolution and its mutation has an adverse effect on
splicing site and protein synthesis. Mutation taster database uses a
third party splice site prediction online software, called NNsplice to
analyze possible changes in splice sites. According to this analysis,
this mutation is likely to disturb normal splicing by omitting donor
site of intron 3 (exon-intron border: wt:CAAG/gtga, mu: CAGG/gtga) and
results in sequence motif lost.
Protein details of UniProt database revealed that this amino acid is
located in NC1 non-collagenous domain which is responsible for the
attachment of anchoring fibrils at amino terminal of collagen VII.
Another splicing site point mutation (COL7A1 , NM_000094: c.682+1
G>A) was identified in family IV which was previously
reported as compound heterozygous with p.R578X mutation. Additionally, a
compound heterozygous 682+1G>C and GS mutation showed
severe phenotypes which indicates expression reduction due to splice
site mutation and exon 5 omission (4, 25).
Point mutation in highly conserved nucleotides such as GT at 5’ end and
AG dinucleotide at 3’ end of an intron have a detrimental effect on RNA
splicing with various deleterious effects on transcription, including
exon skipping, intron retention or frameshift mutation which might
result in in-frame PTC and NMD (24).
Non frameshift deletion and frameshift insertion (COL7A1 ,
NM_000094:c.180-182del.p.60-61del & c.179-180insGAAA,p.F60Fs) were
detected in family III. The identified mutation in exon 2 ofCOL7A1 seems pathogenic in this extremely gene.
The conservation values of the nucleotides in this region is 1 in
phastcons and 3.85 in phylop. Phastcon online software evaluate the
given nucleotide conservation based on the multiple sequence alignment
across genome sequences of 46 different species (The values vary between
0 and 1). The Values close to 1 reflect the high probability of
conservation. Phylop measure conservation as well as acceleration of the
evolution. Positive scores revealed conserved sites and negative scores
predict fast-evolving sites.
The wild type amino acid of stop codon is at 2945 while the mutant one
is at amino acid 88. Therefore, frameshift mutation could indirectly
lead to in-frame PTC and ultimately produces truncated proteins that
might have the potential to interfere the function of normal protein by
dominant negative effect. In this family the mutation give rise to
extremely short protein and NMD seems to be crucial to protect cells.
The identified mutations improve scientific knowledge of the EB
causative mutations, which facilitates the EB classification to consider
effective treatment and appropriate ways to take care of patients.
Moreover, these genetic data will help to investigate the frequency of
these mutations in other affected Iranian families and perform genetic
counselling accurately.