Molecular Genetics Analysis Of Xeroderma Pigmentosum And Hereditary Gingival Fibromatosis

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease which is characterized by extreme sensitivity to sunlight, resulting in sunburn, pigmentation in exposed skin, and disorder in eyes and nervous system. The highest risk of this disease is the cancers with high incidence and recurrence rate. XP-V is a very common subtype of XP disorder and that has similar sun-damage phenotypes with other subtypes. The only difference between XP-V and other subtypes is the unaffected nervous system. Though XP-V has a high incidence compared other subtypes, a deepgoing clinical research still have been lacked. Some XP-V patients without tumor maybe benefits from the young age, but it doesn’t mean they will lose the possibility of cancers. Most XP-V tumors are in oral maxillofacial region and this may result from the more UV irradiation in this region. And this region has many important organs such as eyes, nose, lips and ears, all of which had tumor reports. But no research was about the distriution of tumors in this region.XP disease has eight subtypes which have different defective genes from each other and separately namely XP-A, XP-B, XP-C, XP-D, XP-E, XP-F, XP-G and XP-V. The affected genes of other subtypes are responsible for Nucleotide excision repair (NER) in DNA repairing process. When they are defective, DNA sun-damaged production cannot be cleaned because of unusual NER, and which will cause XP phenotypes in the end. While the gene POLE responsible for XP-V encodes polymerase ηand plays key roles in translesion DNA synthesis (TLS). When polymerase ηare abnormal and cannot carry out TLS as usual, the accumulation of DNA broken sites will finally disrupt cell genome and induce tumor. Human POLH is located on chromosome6p21.1. The longest transcript of this gene in Ensembl database has11exons. It translates a peptide of713amino acids (aa). And the N-terminal511aa are necessary for the activity of the polymerase while the C-terminal70aa are responsible for nuclear localization, the amino acids in middle region have not been studied clearly in their function. Whatever in N-terminal, C-terminal or middle region, many mutations have been reported by now. But no research involved the distribution of mutation or whether some mutation sites with high incidence exist.Since POLH is the only affected gene for XP-V, almost every research for XP-V tumor etiology was limited to finding POLH mutation. No research has demonstrated other factors could promote XP-V tumor formation. It has been confirmed polymerases ι, κ and ζ are also responsible for TLS, but these polymerases have error-prone feature, while polymerase η is the only error-free polymerase to bypass thymine-thymine cyclobutane pyrimidine dimer (TT-CPD), a UV-induced DNA damage product. And another polymerase, polymerase θ, has the same function of polymerase η to generate mutations A/T during somatic hypermutation of Ig genes. Since these polymerases all have similar function as polymerase η, they may have unusual expression when polymerase η is defective. So it’s very valuable to research the change expression of these polymerases in XP-V cells, and the relationship between such polymerases and XP-V tumor formation.Objects:1. Ascertain XP-V patient in clinic, summarize past XP-V tumor reports and discuss the distribution feature of XP-V tumor in oral maxillofacial region.2. Find the mutation in POLH from XP-V patient.3. Construct the vector with mutant sequence and transfect it into cell lines, observe whether it has normal function in cell lines.4. Culture XP-V tumor cells and normal control cells, XP-V cell lines and HSF cell line, HeLa cells with and without POLH knocked down. Meanwhile construct the UV-damaged model of these cells, observe before and after UV irradiation, whether changed expression of polymerases ι,κ,θ andζ accompany with the unusual expression of polymerase η.Methods:1. After getting the informed consent, we inquired the patient history, collected6ml peripheral blood from the patient and normal control people to extract DNA and construct immortalizational B lymphocyte. We also cultured the cells from lip tumor tissue and normal control tissue, normal control was from normal lip tissue of children undergoing cleft lip repair surgery.2. Screened POLH sequence of patient by direct sequencing to find the mutation. Summarize all reported mutation to find distribution feature of mutation sites.3. Separately cloned mutant and normal sequence into pEYFP-C1vector, then transfected these vector into cell lines to observe whether the polymerase encoded by mutant sequence is defective.4. Used Real-time PCR and western blot methods to test expression of polymerases η,ι, κ,θ and ζ in XP-V cells and normal control cells, and HeLa cells with and without POLH knocked down. This observation was also done in those cells suffered UV irradiation.Results:1. The XP-V patient in our research has a typical sun-damaged phenotype. Analysis of XP-V reports indicated in our country, nose has the highest incidence of tumor in oral maxillofacial region. And squamous cell carcinoma is the most common type in this region.2. After sequencing all POLH exons, mutation:g.IVS4+7307-IVS9+1414Del (c.491-1074Del), were confirmed. The mutation sites have no obvious distribution feature in coding region.3. A truncation encoded by mutant sequence can’t locate into nuclei as normal polymer ase.4. The dose of irradiation in our experiment could cause obvious UV-toxicity. Low expression of polymerases κ,θ and ζ could be found in XP-V cells and this may decrease TLS efficiency. But after UV irradiation, these polymerases changed higher expression in XP-V cells and this may increase mismatch rate in DNA replication. In HeLa cells, whatever suffered UV irradiation or not, those genes all showed lower expression when POLH was knocked down.Discussion1. The new mutation in our research translates a truncation of polymerase η with only164amino acids intact and loss of key regions responsible for both activity and nuclear localization.2. Polymerases κ,θ and ζ will decrease their expression when polymerase η is defective. In XP-V cells, these unusual polymerases could increase instability of genome, which also accelerate the process of tumor formation. Hereditary gingival fibromatosis (HGF) is an autosomal dominant disease, which developed as slowly progressive enlargement of gingiva. And a few cases also show autosomal recessive mode. It can be divided into two types, non-syndromic HGF and syndromic HGF. Besides gingival enlargement, syndromic HGF has other phenotypes such as bushy hair or unusual nervous system whereas non-syndromic HGF doesn’t. And most HGF cases in stomatology hospital are non-syndromic HGF form.The sedimentation of extracellular matrixc (ECM) is responsible for gingival enlargement in HGF. Most factors such as TGF-β1and MMPs have been demonstrated to have influence on it. The imbalance of these factors in cells will accelerate gingival ECM fibrosis. Although many risk factors have been reported, the main pathogenic gene of non-syndromic HGF has not been found. SOS1is the only confirmed gene causing this disease, but only one family with its mutation has been reported. There were four candidate genes locus responsible for non-syndromic HGF, which were separately named GINGF1to4. But no definite pathogenic gene in them has been found except SOS1in GINGF1. And further evidence showing genetic heterogeneity of HGF indicated more difficulty on the candidate gene research.Family resource is very important for hereditary disease research. Collection of disease sample is the first step in research. In our previous study, we have collected more than a dozen HGF families and used one of them to locate GINGF3in genomic DNA. The way of location is linkage analysis, but this way only supplied a region in chromosome and could not locate an exact candidate gene. So much work is still needed to find the pathogenic gene in GINGF3, and this research will try to do it. In addition, we still tried to collect more new HGF family resource, which may be very useful in the future study.The region of GINGF3is very large and includes more than100genes. The traditional PCR sequencing technology is not suitable for so many genes. Sequencing of this large region needs a technology with high efficiency and high-throughput screening. As a new technology, exome sequencing array is very fit for those requirements. By this technology, one sequencing scanning could sequence almost whole coding region in genomic DNA. To avoid false positive and false negative, it can ensure dozens of repeated sequencing for one covered base. So this technology is very adapted for candidate gene search in GINGF3.Gene expression chip is usually used to find the difference of genes expression between disease sample and normal sample. So we can use it to find the genes with unusual expression in HGF gingival tissue. Then sequence the candidate unusual genes in GINGF3and ascertain the pathogenic gene.So, this research tried to find candidate genes in GINGF3by exome sequencing array and expression chip. Then sequence the candidate genes and verify the mutation of HGF.Objects:1. Collecting more new HGF families to prepare resource for future research.2. Find the mutation in GINGF3by exome sequencing array.3. Find the pathogenic gene in GINGF3by expression chip.Methods:1. Make a detailed record of each new collected HGF patient. Extract DNA and construct immortalizational B lymphocyte from blood samples of family members. Collect the hyperplastic gingiva from patient to culture HGF fibroblasts, and collect control gingiva from normal people when their opsigenes extraction.2. Choose two patients DNA from GINGF3family, three patients DNA and one normal control DNA from another family, to sequence their coding region by exome sequencing array. Verify the candidate variations in other family members and rule out the SNP possibility by100normal people.3. Choose two patients hyperplastic gingiva from GINGF3family as an abnormal group, use three normal gingva samples as a control group. Compare the difference of genes expression between these two groups. Find the GINGF3genes with unusual expression in hyperplastic gingiva, and sequence these genes to search mutation.Results:1. Three new HGF families were collected. And some samples DNA, immortalizational B lymphocyte, gingival fibroblasts were successfully extracted.2. As the result of exome sequencing, a variation of XDH in GIGNF3family and a variation of WDR91in another HGF family were considered as candidate mutations. But in the following experiment, they were all demonstrated as unreported SNP in normal people.3. The result of expression chip showed only one gene, KCNK3, has insignificant higher expression in HGF gingiva. And all exons of this gene have been demonstrated to have no mutation by PCR sequencing.Conclusions:1. Big difference of expressivity may be observed in one HGF family. The unconspicuous phenotype may be a serious hindrance to make a definite diagnosis for family member.2. The result of exome sequencing array didn’t show any mutations of HGF. The mutant site may be in the uncovered sequencing region or unknown gene region.3. No mutation was successfully found by expression chip. Considering mRNA level, no gene in GINGF3region has obvious unusual expression in HGF hyperplastic gingiva.