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A glycine substitution in the COL7A1 gene causes mild RDEB in a Pakistani family

European Journal of Dermatology. Volume 16, Number 6, 615-9, November-December 2006, Genes and skin

DOI : 10.1684/ejd.2006.0007

Summary

Author(s) : Liv Kraemer, Muhammad Wajid, Angela M Christiano , Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, VC-1526, New York, New York 10032 Fax: (+1) 212-305-7391, Department of Genetics & Development, Columbia University, New York, NY, USA.

Summary : Pathogenic glycine substitutions can cause destabilization of the triple helix and a diverse range of heritable connective tissue disorders, dependent on the collagen gene in which the mutation occurs. Mutations in the type VII collagen gene (COL7A1) cause an inherited mechanobullous skin disease known as dystrophic epidermolysis bullosa (DEB). Typically, the dominant forms (DDEB) result from glycine substitutions within COL7A1, whereas other glycine mutations are ‘silent’ in the heterozygous state and produce disease only when they are homozygous. We studied three affected individuals from a large inbred Pakistani family with a history of skin fragility and scarring indicative of dystrophic EB. We identified a new glycine substitution within the collagenous region in exon 94 of the COL7A1 gene. This mutation, designated G2422V, resulted in a glycine (GGA) to valine (GTA) substitution presumably causing a destabilization of the protein by interrupting the Gly-X-Y repeats. This finding expands the allelic series of COL7A1 mutations underlying mild recessive dystrophic epidermolysis bullosa (RDEB) and sheds further light upon regions of the type VII collagen triple helix that are tolerant of heterozygous glycine substitutions.

Keywords : bullous disease, glycine substitution, mutation, recessive dystrophic epidermolysis bullosa, triple helix, type VII collagen gene (COL7A1)

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Auteur(s) : Liv Kraemer¹, Muhammad Wajid¹, Angela M Christiano^1,2

¹Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, VC-1526, New York, New York 10032 Fax: (+1) 212-305-7391
²Department of Genetics & Development, Columbia University, New York, NY, USA

accepté le 14 Juillet 2006

Epidermolysis bullosa (EB) is a heterogeneous group of inherited mechanobullous disorders characterized by fragility of the skin and blistering [1]. According to the different levels of blister formation within the skin, epidermolysis bullosa is categorized in three different groups: epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB) and dystrophic epidermolysis bullosa (DEB). Whereas blisters in EBS are located in the keratinocytes of the epidermis and those in JEB form within the basement membrane zone, the tissue separation of DEB occurs on the dermal side of the basal membrane zone, in the region of the anchoring fibrils [2]. The reasons for the different levels of the skin separation are now explained at the genetic level since mutations have been identified in ten distinct genes whose proteins are expressed at the dermal-epidermal junction [3]. Consequently the different variants of EB are now defined by the site of their molecular lesion as well as their clinical findings [4].The dystrophic forms of epidermolysis bullosa can be inherited either in an autosomal dominant or recessive manner [5]. The characteristic ultrastructural findings of the cutaneous basement membrane zone (BMZ) of DEB demonstrate various degrees of morphological abnormalities of the anchoring fibrils [6], which maintain adhesion of the epidermis to the dermis [7], and range from subtle changes to complete absence [8]. These consist predominantly of the type VII collagen gene [9,10], encoded by a gene (COL7A1), which is localized on chromosome 3p21.3 [11, 12]. The type VII collagen fibril is composed of three identical alpha collagen chains and is characterized by Gly–X–Y repeat sequences [13], containing several interruptions [14], including a 39-amino acid non-collagenous segment in the middle of the collagenous domain, called the “hinge” region.Within the two non-collagenous domains (NC1, NC2) and the helical domain of COL7A1 more than 200 distinct mutations have been identified in DEB patients [15,16]. In most of the cases, the mutations have been reported to be specific to individual families or populations [17].The majority of glycine substitutions in COL7A1 typically underlie dominant dystrophic epidermolysis bullosa (DDEB). In contrast, mutations in the recessive DEB (RDEB) phenotypes tend to be more severe [18]. In Hallopeau Siemens RDEB, the mutations often cause premature termination codons in both COL7A1 alleles, resulting in the complete absence of anchoring fibrils. In mild RDEB, typically at least one other molecular mechanism was identified that involves recessive missense mutations resulting in decreased stability and/ or altered function of the synthesis of type VII collagen [19]. Paradoxically, however, recessively inherited glycine substitutions have also been reported in COL7A1, that are silent in heterozygous carriers.In this study, we describe a homozygous glycine substitution mutation in a patient of a large inbred family of Pakistani origin with mild recessive DEB inherited from both parents who are heterozygous, clinically unaffected carriers of this mutation.

Materials and methods

Clinical information

The parents are second/ third-degree relatives of Pakistani origin. There have been no known skin diseases in this family before. The proband, a twelve year-old female (V-1), developed skin changes at the age of 16 days and shortly thereafter, scarring and erosions as well as feet nail deformities that increased during adolescence. The blisters are found mainly in the forearm region and especially around the patella, tibia, fibula and the foot ( (figure 1B) ). Her teeth and hair are normal and the clinical examination shows no abnormalities. There are two affected younger brothers (V-2, V-3), eight and four years old, with the same appearance. The parents and the other family members are unaffected. The diagnosis of DEB was established on the basis of typical skin symptoms such as blisters on trauma exposed body sites such as hands, knees and feet together with characteristic nail changes and severe scarring.

DNA analyses

Blood samples were collected from the family, including three affected and two unaffected siblings, and her unaffected parents of Pakistani origin, in tubes containing EDTA. All individuals provided informed consent for inclusion in the study, in accordance with guidelines set forth by the institutional review board. Genomic DNA was isolated according to standard techniques using Pure Gene DNA Isolation Kit (Gentra Systems, Minneapolis, MN) [20].

PCR amplification and mutation screening

A mutation detection strategy for the COL7A1 gene was developed based on PCR amplification of all 118 coding exons using 72 primer pairs placed on flanking intronic sequences, which were designed previously [21].

For mutation screening, all exons and splice junctions were PCR-amplified from genomic DNA. PCR products were sequenced in an ABI Prism 310 automated sequencer, using the ABI Prism Big Dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA), following purification in Centriflex gel filtration cartridges (Edge Biosystems, Gaitherburg, MD). Mutations were identified by visual inspection and comparison with control sequences generated from unrelated, unaffected individuals of Pakistani origin.

Mismatch PCR

To confirm the mutation identified in exon 94 of the patient and for studying the inheritance of the mutation in the family members and control individuals of Pakistani origin a mismatch PCR strategy was used. The missense mutation G2422V was confirmed by digestion of the corresponding PCR products with the restriction endonuclease Rsa1 (recognition site 5’GT/AC3’) (New England Biolabs, Beverly, MA), respectively [22]. A reverse mismatched primer (5′-AGCCCCTCACCCGCTGT-3′) was used to introduce an Rsa1 restriction site. Finally, the PCR product was run on a 1 % agarose gel and visualized by ethidium bromide staining ( (figure 2B) ).

Results

By examining the molecular basis of a large inbred family of Pakistani origin, we identified a new glycine substitution within the collagenous region in exon 94 of the COL7A1 gene, which is clinically silent in the heterozygous carriers but causes mild recessive dystrophic epidermolysis bullosa (RDEB) when homozygous in affected individuals. The mutation resides 1084bp upstream from the non-collagenous region 2 (NC-2) and consisted of a G-to-T transversion at nucleotide position 7265 of the COL7A1 cDNA (numbered according to GenBank L23982). To our knowledge, this nucleotide substitution represents a previously unreported missense mutation and results in the conversion of a glycine codon (GGA) to a codon for valine (GTA) at position 2422, designated G2422V ( (figure 2A) ). Automated sequence analysis of this PCR product of all family members revealed the same sequence variant in the homozygous state in the patient (V-1), her two affected brothers (V-2, V-3) and in the heterozygous state in her unaffected sister (V-5) and her parents (IV-1, IV-2).

To confirm the sequence variation in exon 94, restriction digestion was performed after using a mismatched PCR strategy with the enzyme Rsa1. The mutation creates a restriction site for Rsa1 on the mutant allele, and after digestion of the PCR product containing exon 94, the proband and her two affected brothers revealed only the digested band of 163 bp, indicative of the mutation ( (figure 2B) ). Digestion of the PCR product containing exon 94 in an unaffected brother (V-4) and in 50 unaffected, unrelated Pakistani control individuals did not show the restriction pattern indicative of the mutant allele, revealing that the sequence variant is not a common polymorphism in this population. The patient’s parents (IV-1, IV-2) and her unaffected sister (V-5) show both fragments after the endonuclease digestion process, demonstrating the heterozygous state of the mutation.

The patient was shown to be homozygous for this mutation G2422V confirming the presumed clinical diagnosis of dystrophic epidermolysis bullosa. DNA analysis in the parents revealed that both carried the mutation in the heterozygous state. We conclude that this glycine substitution is a recessive mutation, as demonstrated by the absence of clinical symptoms of EB in the carriers of the mutation.

Discussion

For all collagens, the repeating sequence pattern (Gly-X-Y)_n, in which glycine is present in every third position, is highly conserved during evolution and essential for the molecular structure-function of these proteins [23]. If the mutation results in a glycine substitution within the collagenous domain, which breaks the Gly-X-Y repeat-pattern, the result is a disruption of the triple helix and a discontinuity in the register of the helix [24]. Therefore glycine substitutions in the triple helical domain of the different collagens lead to a wide spectrum of heritable connective tissue diseases. Glycine replacements in collagen type I-IV and VII for example have been shown to result in osteogenesis imperfecta, osteochondrodysplasia, Ehlers-Danlos syndrome type IV, Alport Syndrome and dystrophic epidermolysis bullosa, respectively [25-27].

The Pakistani patient described herein represents a new case of RDEB in which a mutation in exon 94 was found within the collagenous subdomains that form the collagen VII triple helix, resulting in a disruption of the typical glycine-X-Y amino acid sequence [28]. This mutation is clinically silent in the heterozygous carriers but causes mild recessive dystrophic epidermolysis bullosa (RDEB) when homozygous in affected individuals. This glycine substitution, designated G2422V, resulted in the conversion of a glycine codon (GGA) to a codon for valine (GTA) and presumably causes destabilization of the protein. According to Persikow et al. the possibility of the destabilization of the triple helix by the replacement of glycine to valine is relatively high [29]. With this mutation, the “GTA” codon could have a theoretical possibility of creating a cryptic donor splice site. However, in silico analysis and the appearance of the relatively mild phenotype suggest, that in fact it does not.

From the over 200 known mutations in the COL7A1 gene, one third of them result in a glycine substitution, among which dominantly and recessively inherited glycine mutations are almost evenly distributed [30]. Among other collagen genes, a silent glycine substitution mutation has been identified in COL4A4 causing Alport syndrome [31] and in COL11A2 resulting in chondrodysplasia [32]. The reasons why certain glycine substitutions remain clinically silent when combined with a normal allelic product are uncertain at this point. It was assumed that one possible reason might be the location of the substituted glycine within the collagen genes and therefore the extent of abnormal folding of the protein or disruption of the helix [33].

This mutation, located in the collagenous segment, resides 1084bp upstream of the non-collagenous region 2 (NC-2). It is surrounded by other silent glycine mutations, one of which located 27 amino acids upstream in the same exon 94 (G2395D), another in exon 95 (G2569R), and is flanked by dominantly inherited glycine mutations in exon 93 and 105. In contrast to dominantly inherited glycine substitutions, which tend to be located near the main non-collagenous hinge region of the triple helix, which provides flexibility to the collagen [34], silent glycine substitutions seem to be position independent.

One of the consequences of basic research on EB is the identification of specific mutations in the COL7A1 gene as a part of an expanding database, in which diagnoses can be improved and a classification with prognostic implications can be refined [35]. Especially for distinguishing “sporadic” dominant versus mild recessive DEB [36], which is not discernable just by clinical examination, histopathology, immunohistochemical or ultrastructural analysis [37], mutation analysis is the only method by which to determine the correct pattern of inheritance [38]. DNA-based prenatal testing of families at risk for recurrence is another benefit of this process. In the past, patients with mild RDEB were counselled as ‘de novo’ dominant mutations [39]. Molecular analysis however, has revealed exactly the opposite, that mild RDEB is more common and true ‘de novo’ DDEB is less frequently observed [40].

In homozygous RDEB, affected individuals carry mutations on both alleles of the COL7A1 gene, usually leading to a defect with a more severe phenotype. In the first year of life, our proband showed a pronounced blistering with subsequent development of typical scarring in the trauma-exposed areas of the body. In accordance with her mutation, she does not show any other abnormalities typical of severe RDEB such as syndactyly or excessive scarring [41]. Despite the fact that the most common cause of death, in patients with recessive inherited EB who survive childhood, is a metastatic squamous cell carcinoma [42], we do not anticipate this in our proband.

This new substitution expands the allelic series of COL7A1 mutations underlying mild RDEB, and sheds further light upon regions of the type VII collagen triple helix that are tolerant of glycine substitutions.

Acknowledgements

We are especially grateful to the patients and their families for their interest in this study. We highly appreciate the skillful help and excellent technical assistance of Ha Mut Lam. We thank Prof. John McGrath, Department of Cell and Molecular Pathology, John’s Institute of Dermatology, The Guy’s, King’s College and St. Thomas Hospitals’ School of Medicine, St Thomas Hospital, London UK, for helpful discussions. This study was supported by USPHS NIH grant from NIH/NIAMS RO1 AR43602 and by a fellowship within the Postdoc-Programme of the German Academic Exchange Service (DAAD).

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