| BackgroundThe Human Genome Project (HGP) was officially started in October1990to2003, which finished mapping the human genome draft, discovered the genetic information that contained to the human life, enriched the number of genetic markers, promoted the development of genomic technology, and plans to continuous study on the all genes that owned by human beings, especially in the genes structure and function that related to the diseases, and the relationship between variety gene mutations and diseases, further clarify the pathogenesis of human genetic diseases, and eventually realize the gene diagnosis and gene therapy of the genetic diseases.In recent years, most experts have made a great achievements in the skin genetic diseases. The scientific research results were published in the top international journals, the conclusions has attracted a great attention to the genetics community in the world, which provides the scientific basis for gene research results with independent intellectual property rights in our country. For the vast territory, large population, ethnic diversity and rich genetic resources, which provides favorable conditions for the study of hereditary skin diseases. Therefore, the collection, preservation and utilization to the rich genetic resources, carrying out the genetic skin disease research in China, which is not only beneficial to improve the domestic understanding and screening level of genetic diseases, but also advantageous to rich the genetic mutant gene mapping in the world.Epidermolysis Bullosa is a group of hereditary bullosa skin disease, express in autosomal dominant (AD) or autosomal recessive (AR) genetic, characterized by damage on the skin or mucous membrane by minor trauma can cause blistering, sometimes referred to as "mechanical bullosa skin diseases". In June,2008, the new classification standard was released officially, according to the standard of the blisters’ position in the skin that identified by transmission electron microscope, EB is divided into Epidermolysis Bullosa Simplex (EBS), Junctional Epidermolysis Bullosa (JEB), Dystrophic Epidermolysis Bullosa (DEB) and Kindler syndrome clinical types, each type also includes different subtypes. EBS cleft occurred in the basal keratinocytes, most of the EBS are caused by the gene mutations of keratin5(Kertin5, K5, KRT5) or keratin14(Kertin14, K14, KRT14), a few of them are caused by gene mutation of net protein, desmoplakin, plakophilin1and a6β4integrin; JEB cleft occurred mainly in the transparent plate, is caused by abnormality of lamina332, α6β4integrin or type XVII collagen; DEB, with separation under the lamina densa, is caused by the defect in collagen VII (Type VII collagen, COL7A1). Kindler syndrome skin separation can occur in the epidermis, junction or sub-layer of densa board, consequently, it cannot be classified as EBS, JEB or DEB in any types. The pathogenic gene is caused by mutation in Fermitin family homologous Protein1.EBS is the first human genetic vesicular disease that described from molecular level, it is also the first human keratin (K) disease, is a most common clinical type of EB, the morbidity is about1:30000-1:50000. According to the position of the skin separation occurs, the EBS can be divided into the basal layer upper side subtype and basal layer subtype, different subtype also includes a lot varieties diseases. The other Sporadic type of EBS is the most common type, express in the autosomal dominant inheritance, with complete penetrance, morbidity of1/500000, called Koebner (EBS-K) type. The clinical features were blister widespread and occurred in hand joint, elbow, knee, foot and other easy to repeated position, characterized of Nissl negative, the nails and mucosa are insensitive to it. Infants usually infected after birth a few days, but it would be better a few months later, however, it maybe relapse at the beginning of crawling and childhood, a parts of patients would have the blister at long term and widespread occurrence. It will be severed during the summer, and eased a little during the winter, it have a few damage, and does not cause serious adermotrophia. It also will be dissipated during the treatment, the patients who have this disease have the general development and normal health status. The pathogenic gene is keratin5and keratin14. K5gene is belongs to a type II keratin family (GenBank no:NM000424), chromosome is located in12q11-ql3, contains9exons; K14gene is belongs to a type I keratin family (GenBank no:NM000526), chromosome is located in the17q12-q21, contains8exons. In recent years, about214of K5and223of K14gene mutations were found in different EBS patients (http://www.interfil.org). According to the literature review, most K5and K14gene mutation with EBS patients are occurred in H1region of the molecular head segment (only in K5gene),1A and2B region of the rod district, and the L1-2segment of1A and2B connecting region, we also found that the clinical manifestations differences caused by different gene mutation region, which lead to the different clinical subtypes. Many studies have confirmed there is a good relationship between K5and K14mutation region and the clinical manifestations, proposed two mutation "hot" areas for the keratin, they are located in the1A starting area and2B end area of two highly conserved α helical rod district, called helical initiation position (HIP) and helical terminal position (HTP), and they are connected with head position and tail position, respectively, which is an important structure of keratin intermediate filament assembly extend. Any sequence changes of this structure can damage the functions of the proteins, leading to severe EBS-DM. The gene mutation occurs in non conservative connected region L1-2can lead to other general EBS and EBS-loc.DEB is one kind of EB with a scar, blisters were located in deep tissue of the skin, under the density board of the upper dermis. DEB can be divided into dominant (DDEB) and recessive (RDEB) types. DDEB-Pt is a rare clinical DEB subtype. Kuske (1946) reported a DEB case with the itching blisters, atrophy and scar in the shins anterior position, called as the shins anterior DEB. It can occurs at birth, may also be in infancy or childhood, mainly occurs in the shins anterior, forearm, a few occurs on the trunk. The patients with this disease have a obviously itching, visible scar, milia and nail dystrophy. Its damage for the adults showed lichenoid patch. The disease is a chronic progressive, and will be worse after the puberty.The DDEB-Pt pathopoiesis gene is a type Ⅶ collagen (COL7A1). Type Ⅶ collagen is the component of anchoring fibrils, it combined with the anchor spot, and interweave with the leather fiber, it plays a very important role in the structure stability maintenance of the dermal epidermal junction. Collagen type Ⅶ is a homotrimer consist of three identical αl chains, each α1polypeptide chain containing central collagenous Tri-helix region, the non collagenous amino terminal and carboxyl terminal were located in its two sides. The repetitive Gly-X-Y sequence consists a Tri-helix structure position in the collagen structural region. Type Ⅶ collagen gene (COL7A1) located in3p21.1, consisting of118exons (Genbank No:L23982), is a gene with most exon reported until now. According to the statistics, more than730COL7A1gene mutations point were reported until now (http://www.col7.info). The most of DDEB mutations are glycine substitution mutation (a base point mutation caused glycine substitution by other amino acids). While RDEB is usually mutated with both two homologous chromosomes mutations, its mutation point changes greatly, usually in the following ways:PTC (Premature Termination Codon)/PTC, missense mutation/PTC, missense mutation/missense mutations, PTC/splice site mutations.This study focused on the one Chinese Epidermolysis Bullosa Simplex patient (EBS, gen-nonDM) and one shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient, surveyed all of clinical information and detected the gene mutations of two patients, one K14gene and one COL7A1gene were mutated, the results showed that K14gene missense mutation of c.280G>A(p.Ala94Thr), while COL7A1genes glycine substitution mutation of c.G6109A(p.Gly2037Arg). We also confirmed that these2mutations are pathogenic mutations, this result is first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it’s need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Sporadic Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy.ObjectiveDetermine the gene mutation in the patients with Epidermolysis Bullosa Simplex(EBS, gen-nonDM) and shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt), understand the relationship between the clinical phenotype and genotypes of EBS and DEB, enrich the gene mutation data; further understanding the pathogenesis of EB,provide an effective information for the future clinical diagnosis, prenatal diagnosis, gene disease therapy.MethodsCollected the blood samples of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient,shins anterior Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) Sporadic patient and their two families’ members and100healthy controls person, extracted the genomic DNA in peripheral venous blood using modified salting out method, conducted the PCR amplification sequence analysis for all exons of the gene coding region and exon intron junction sequences of6pairs primers of K5and118exons of the gene coding region and exon intron junction sequences of72pairs primers located in COL7A1gene.ResultsThis study examined one Epidermolysis Bullosa Simplex (EBS, gen-nonDM) patient and one shins anterior Sporadic Dominant Dystrophic Epidermolysis Bullosa (DDEB-Pt) patient. The patient of cases one is a5year old female, the body appears widely Epidermolysis Bullosa at her born, form a lot of different sizes blister after scratching, trauma or stress, the blister with some clear contents inside are easy to break, and also easy to healing. The friction parts of the skin recurrent blister and bullae, unequal in size, these blister and bullae can own broken, and also can self healing, no scars after healing. The disease repeated again and again. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermal hyperkeratosis, sub-epidermal blistering, inflammatory cell infiltration. The patient of case2is a11years old male, with the bean size blister in the second joint ventral of right index finger at his born, there is scar left after blister wall rupture, subsequently, bean size papula and blister appears in the bilateral ankle, appears the hypertrophic scar in parts of the skin after blister burst. The patient was treated in many hospitals, given vitamin E, traditional Chinese medicine (composition is unknown) oral and hormone ointment for the treatment, the rash did not control, and extended to the scalp, trunk and limbs, the scar increased, hypertrophy, rash especially in the shank part side and the elbow are very heavy. Since2011, the patient have the chronic urticaria, the rash itching is more severe, the blisters were occurred after scratching, collision or friction, and scar thickening, extension, texture tougher day by day. The patient has no similar family history of the disease, the parents are not consanguineous marriage. Histopathology showed:epidermis hyperkeratosis, formed several multiple angle plug, irregular acanthosis hyperplasia, the bullous formed under the visible epidermal, individual acantholysis shedding to the blister inside, dermal papilla edema, superficial dermal capillary hyperplasia and dilatation, blister and perivascular inflammatory cell infiltration, including lymphocytes, eosinophilic granulocyte and neutrophils, dermal collagen fiber hyperplasia. Using the genomic DNA of two patients as template, all18pairs primers in case one and72pairs primers in case two were amplified in different condition, all amplify products were purified and sequenced, then compared with K14and COL7A1gene sequence that published in the Ensembl website, the results showed, in case one, guanine (G) in280loci of K14gene exon1replaced by adenine (A)(c.280G>A), which resulted in the94th codon in the head position changed from GCT to ACT, and leads to the alanine (Ala) in the head position of K14gene mutated to the threonine (Thr), namely c.280G>A(p.Ala94Thr) missense mutation, the same mutation was not found in the her parents and100normal control person; in case two, guanine (G) in6109loci of COL7A1gene exon73replaced by adenine (A), which resulted in the2037th codon of Tri-helix region changed from GCT to ACT, encoding amino acid changed from glycine (Gly) to arginine (Arg), namely c.G6109A(p.Gly2037Arg) glycine substitution mutation, the same mutation was not found in the his parents and100normal control person.ConclusionUsing the molecular biological techniques, such as the genomic DNA extraction, PCR amplification and direct sequencing, detected the gene mutation of the patients who are one of Epidermolysis Bullosa Simplex (EBS, gen-nonDM) and another of Sporadic Dystrophic Epidermolysis Bullosa (DDEB-Pt), the results showed that one is K14genes missense mutation c.280G>A (p.Ala94Thr) and another is COL7A1genes glycine substitution mutation c.G6109A (p.Gly2037Arg). We are also confirmed that all these2mutations are pathogenic mutation, this result is a first report in domestic and abroad by us. Whether Sporadic patients with DDEB-Pt are de novo mutation or not, it’s need further study and discussion in the future. The study extends the genetic and clinical database of Epidermolysis Bullosa Simplex and Dystrophic Epidermolysis Bullosa for Chinese person, laid the foundation for genetic counseling, prenatal diagnosis and gene therapy. |
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