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Epidermolysis Bullosa: Mutations of Type VII Collagen Gene

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A novel deletion mutation and two recurrent substitutions on type VII collagen gene in seven Iranian patients with epidermolysis bullosa

  • Armita Kakavand Hamidi, Mohammad Moghaddam, Nasim Hatamnejadian, Bahar Sedaghati Khayat, Ahmad Ebrahimi


Epidermolysis bullosa is one of the most important series of mechano-bullous heritable skin disorders which is categorized into four major types according to the layer that bullae forms within basement membrane zone. In dystrophic form of the disease, blisters are made in the sublamina densa zone, at the level of type VII collagen protein which produce anchoring fibrils. Type VII collagen gene is the only responsible gene for this form. The aim of this study was to survey causative mutations of type VII collagen gene among affected Iranian patients with epidermolysis bullosa. For this purpose, exons 73-75 were investigated by polymerase chain reaction followed by direct sequencing. In current study, we found three different point mutations in type VII collagen alleles in 7 out of 50 patients. Four patients were homozygous for a new deletion which resulted in frame shift (p.Pro2089fs). Two patients were homozygous for a recurrent glycine substitution (p.G2031S) and one patient was detected with an allele carrying a substitution (p.R2069C). The results emphasized heterogeneity in the type VII collagen gene and will provide a sign for early diagnosis and future study of the disease pathogenesis.

Key words: type VII collagen gene, dystrophic epidermolysis bullosa, mutation


Epidermolysis Bullosa (EB) is a series of mechano-bullous skin disorders in which skin and mucous membrane become very suceptible to fragility and blistering. Variable diagnostic methods such as skin biopsy, clinical examination, hystological experiences have revealed four major subtypes.(1) (a) In the simplex type of EB, the tissue is disrupted at the intraepidermal layer and mutations in genes KRT5 and KRT14 are involved in formation of this type of the disease. (b) Junctional form of EB is as a result of mutations in laminin 5, COL17A1, ITGB4 and ITGA6 genes and tissue separation occurs in lamina lucida.(2) (c) In dystrophic epidermolysis bullosa (DEB), abnormal anchoring fibrils structure, decreased number or lack of them make rupture under the basement membrane zone (sub-lamina densa).(2-3) Both two forms of DEB, dominantly inherited and recessively inherited (DDEB; MIM# 131750 and RDEB; MIM# 226600) have been detected with extremely variable blistering trend. The only gene which has genetic linkage with both RDEB and DDEB is collagen, type VII, alpha- 1 (COL7A1; MIM# 120120). This gene has been mapped to chromosomal region 3p21 and has 118 exons.(2, 4) The protein type VII collagen is made of three identical subunits called α1 (VII) chains. Every α1 (VII) chains includes a 145 kilodalton (KD) zone (central triple helical collagenous region), flanked by a longer non-collagenous (NC-1) domain (exon 1-28) and a smaller carboxy-terminal non-collagenous (NC-2) domain (exon 112-118). The central triple helical collagenous part includes a repeatative Gly-X-Y sequence. Through out the producing anchoring fibrils (AFs), two collagene type VII molecules localize in an antiparallel direction which is fixed by disulfide bonds. Then these dimers assemble in a way that contain the NC-1 domain at both end of these structures and result in AFs’ production.(3) It has been reported that many mutations have trend to cluster in exon 73 (10.74%) and this indicates that this exon represents a section in which mutations normaly affect the function of AFs. Also a common state for mutations which impact exons 73-75 as part of a Gly-X-Y sequence may lead to a more destructive glycine substitution than the other parts of the protein.(4-5) According to importance of exons 73-75 of COL7A1, we inclined to survey exons 73-75 of COL7A1 gene in order to get the picture of targeted exons’ causative mutations (type and prevalence) among Iranian patients with EB. A novel and two recurrent mutations were discovered which resulted in revealing inheritance pattern among the cases under study.


According to salting out method, Genomic DNA was extracted from peripheral blood lymphocytes of 50 patients and used for amplification of COL7A1 gene sequences. Achieving this purpose, two oligonucleotide primers (forward and reverse) were designed around exons 73-75 and their flanking intronic regions. The primer sequences for generation of a 475 base pair (bp) fragment consisting exons 73-75 were as follows: Upstream primer sequence: 5’ TCT GGT AGG TTC CTG CCT GT 3’ and downstream primer: 5’ AAT TCC AGG GTT ATG GCACA 3’. For polymerase chain reaction (PCR) amplification, 500ng of genomic DNA was used as template. A thermo cycler (TECHNE, UK) was employed for amplification. PCR cycles were initiated at 95 ÌŠC for 5 minutes for the first cycle. The PCR setting for the next 30 cycles was denaturing at 95 ÌŠC for 50 seconds, annealing at 62 ÌŠC for 45 seconds, and elongation at 72 ÌŠC for 1 min with a final elongation at 72 ÌŠC for 5 minutes. Amplification buffer was contained of 100 mM MgCl2 (0.5 μl) and 5 U of Taq polymerase (0.2 μl). Final volume of PCR was 5 micro liter. PCR product was applied for electrophoresis through a 1.0% agarose gel and also for gene sequencing in order to diagnose the causative mutations. The mutations were detected using sequence analyzer software and the human mutation data bank (HGMD).(6) Every accepted proposal in Guilan University, Tehran, Iran, should follow ethical rules and welfare, otherwise, it will not be accepted. All patients gave written consent for molecular analysis.


Totally, 14% of causative mutations (3 different mutations in 7 out of 50 probands) were discovered at least in one allele. In 6 probands, mutations were found in both alleles of COL7A1 gene (homozygote) and in one patient a mutation was disclosed in an allele of the gene. Direct sequencing revealed a G to A transitional position 6091 within exon 73 of COL7A1. The mutation that causes a glycine codon (GGC) exchanged into a serin (AGC) is described as p.G2031S. This kind of mutation was detected in two patients. A newly detected mutation in four patienets was a deletion of a C nucleotide at position 6265 within exon 75 of COL7A1. This deletion that consider as p.Pro2089fs, resulted in a frameshift within the collagenous domain of α1 (VII ) chain. Only one patient had p.R2069C mutation that revealed as a C to T transitional position 6205 within exon 74 of COL7A1. This mutation had converted an argenine codon (CGT) into a cysteine codon (TGT). parental consanguinity in 84% of studied families and no record of the disease among other members of 58% of studied families were detected.


In this study, we showed one novel and two discrete recurrent mutation within COL7A1 gene in 7 out of 50 probands from unrelated families. The new detected mutation was a single C deletion (p.Pro2089fs) in both alleles of four patients. The other two recurrent mutations (C.6205C>T & C.6091G>A) were an allele consisted of a transversion in one patient and a Gly substitution in both alleles of two patients respectively. Therefore, Three out of seven detected mutations was missense. All these detected mutations was located in collagenous domain of COL7A1 within exon 73-75.

In current study, the presence of parental consanguinity in 84% of studied families certified the direct relation between parental consanguinity and the probability of mutated alleles inheritated from a common ancester.(7) Hence it is suggested that even relatives with no any signs of a disease do not get married preferably. There was no record of the disease among other members of 58% of studied families. Thus, consider to our results and according to the pedigrees, most of the detected causative mutations were just in the affected member of that family and it happened for the first time. There are lots of investigations which suggest that sequencing analysis of exons 73-75 of COL7A1 gene is able to detect approximately 75% of causative mutations in patients who are suspicious of DDEB (8) and also 95% of cases on the condition of exact diagnosis of DEB.(9) In other words, 10.74% of known mutations in COL7A1 are gathered in exon 73.(4) In despite of most of other surveys in which all 118 exons of COL7A1 gene have been investigated,(5, 8, 10-12) in our study, we examined the sequence of three exons (73-75) in 50 patients with EB (regardless the type of EB) that resulted in detection of 14% of causative mutations at least in one allele. Consequently, this study can be used as the first step in the screening of Iranians with EB, especially, the ones who are suspicious of having DEB.

Substitution in the triple helical domain of type VII collagen, especially in exons 73, 74, and 75 is the cause of catching DDEB in more than 75% of all cases.(8) Mutations p.Gly2043Arg and p.Gly2034Arg are the most common mutations which include more than 50% of dominant mutations among bigest studied group of USA population.(5) The tendency of Gly substitution to accumulating within exons 73-75 of COL7A1 disclose that the place of Gly in each third condition through triple repeat of type VII collagene is critical for triple helical structure stability.(5) These substitutions could lead to anchoring fibrils instability and making them sensitive to mechanical stresses.(4) According to Nordal study,(13) the probands that carry p.G2031S might have RDEB-HS phenotype. Whenever two missense mutations are identified in a proband genomic DNA, his/her parents are obligatory heterozygous carriers of the same mutation and the inheritance pattern is assumed autosomal recessive. This is important because the distinction between autosomal recessive and autosomal dominant is necessary for presenting accurate genetic counselling, and significantly to prognosis the risk of childbirth with the same disorder in next generation.(5)

p.R2069C mutation is a result of cytosine to tymidine transition in nucleotide 6205 in exon 74. So aminoacid argenine is replaced by cystein in codon 2069 of collagen type VII protein in the triple helical collagenous domain. This mutation is reported by Kahofer et al. in 2003, in compound heterozygous status who had RDEB-I phenotype.(14)

The novel and most frequent mutation among detected mutations was a deletion in exon 75 of COL7A1 gene which was found in four unrelated families. This deletion resulted in a frameshift at the amino acids 2089-2093 of type VII collagene within triple helical collagenous domain and changing in the number and order of aminoacids PPGPK in normal protein type VII collagene to PLAP in mutated type VII collagen. This de novo mutation is a homozygous one, although true possitive cases of de novo mutations are inherited dominantly.(4) In this study the detected recessively inherited de novo mutation suggests that parents of these families should be called germline musaisms in order to precise genetic counseling, because the causative mutation might occure in other children of the family. It seems reasonable that we could find new deletion originated from Iranian people which lead to RDEB with regard to other reported mutations which are come from especial family from different countries such as R2814X- 7786delG- R578X originated from Great Britian, 6573+1G>C- E2857X- 5818delC originated from Japan, 425A>G- 8441-14del21- 497insA originated from Italy, C-6527insC originated from Spain, and 2470insG originated from Mexico.(15) As a result, our study imply the variety of mutational events which cause RDEB phenotype and emphasizes the molecular heterogenity of DEB.

In our study, the frequency of detected mutations from high to low rate was belonged to exons 75,73 and 74 respectively.

Other studies that have dealt with the adjacent codons to codon 2089 have revealed some mutations exactly at backward and forward codons.(10, 16-17) This may be due to sensitivity of this location to mutations, especially, deletion mutation. Hence, it could develop a new screening method and underlines importance of further assessment on a larger population of Iranian patients with EB.

In conclusion, one of the things resulting from research like the current one on epidermolysis bullosa is the identification of specific and new mutations in COL7A1 gene. Also, extension of mutations data banks makes an accurate diagnosis and better DEB classification besides other diagnostic methods based on clinical histological features and pedigree pattern. In addition, these kinds of studies are used for disease prediction, presenting better genetic counseling and prenatal diagnosis in families with high risk of the disease recurrence and for choosing appropriate candidates for gene therapy.


We gratefully appreciate participants and their family members.

This study has been supported by skin research center of Shahid Beheshti University and Parseh medical genetic counseling center.


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