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.