Relationship Between Site Of KIT Mutation And Clinical Phenotype On Patients With Piebaldism

Piebaldism （MIM172800） is an autosomal dominant disorder characterized by congenital patches of leukoderma and poliosis, which principally locate on the mid-forehead, ventral chest, abdomen and extremities. The depigmented patches are present from birth and generally keep static in shape and distribution with typical hyperpigmentation or normal pigmentation islands within and at the borders of the depigmented areas. Histopathological examination shows that epidermal melanocytes are usually absent in the affected skin, the result of defective proliferation of embryonic melanoblasts before its migration from the neural crest into the dermis.It has been demonstrated that piebaldism is due to inactivating mutations or deletions of the KIT gene on chromosome4q12or deletions of the SLUG gene on chromosome8q11. Spritz first reported that KIT gene mutation caused human piebaldism. The KIT gene consists of21exons distributed over more than70kb at chromosome4q12. It encodes a976-amino-acid protein, a type III transmembrane receptor tyrosine kinase with an amino-terminal extracellular domain composed of five immunoglobulin-type repeats, a single transmembrane segment, and a cytoplasmic kinase domain comprised of a bipartite tyrosine kinase domain. Binding of SCF to the extracellular domain induces the receptors to form dimers and activates the intracellular tyrosine kinase through the transphosphorylation of specific tyrosine residues, then results in consequent phosphorylation of tyrosine residues both on KIT itself and on various distal proteins in the.KIT-mediated pathway of signal transduction, and ultimately culminates in cell proliferation. In the year of2003Sanchez-Martin discovered that SLUG was a second major gene linked to human piebaldism. The SLUG is a zinc-finger neural crest transcription factor, encoded by the SLUG gene, which is critical for development of melanoblasts during its migration from the neural crest into the dermis.It has been demonstrated that about75percent of patients with piebaldism have mutations on the KIT gene, which encodes the stem cell growth factor （SCF） receptor. Richard discovered that the KIT mutations could be classified into three general groups, each group tending to be associated with different piebaldism phenotypes. The first group of KIT gene mutations consists of missense substitutions that result in amino acid substitutions, all of which are located within the intracellular tyrosine kinase domain. These KIT missense substitutions are all related with relatively severe phenotypes, a consequence of the fact that the KIT kinase is only activated on dimerization of the receptor. In patients heterozygous for KIT missense mutations one-fourth of the KIT receptor dimers contain the abnormal KIT polypeptide, and half of the KIT receptor dimers consist of one normal KIT polypeptide and one abnormal KIT polypeptide. These heterodimers are inactive. Thus, patients heterozygous for these KIT missense mutations have only one-fourth of the normal amount of KIT receptor. In contrast, the second group of KIT mutations completely eliminate the production of KIT protein by the mutant gene, all of which are located within the extracellular tyrosine kinase domain. Patients heterozygous for these mutations express only half of the normal amount of KIT receptor. However, half amount of the KIT receptor is not adequate for KIT dependent signal transduction. These KIT mutations are thus associated with relatively mild phenotypes. The third group of patients with piebaldism exhibit a very variable phenotype, ranging from extremely mild to quite severe usually resulted from frameshift and splice junction mutations which locate on or around the transmembrane region. These mutations would result in premature termination of translation, truncating the nascent KIT polypeptide distally. The truncated KIT receptor can still bind steel factor and form dimers, but negatively inhibiting function of the normal KIT polypeptide. However, both the truncated KIT polypeptides and the incompletely translated KIT mRNA are relatively unstable. Therefore, these mutations reduce KIT receptor function to an amount between one-fourth and half of normal, accounting for the intermediate and highly variable phenotype.The relationship between KIT mutations and piebald phenotypes is very complicated. But in China little of researchs on piebaldism have been carried out, while none of research on the relationship between KIT mutations and piebald phenotypes. It is necessary to do some research to find out the major KIT mutations in China and the relationship between KIT mutations and piebald phenotypes. What is more, it is the only way to search for a method in order to cure the piebaldism through prenatal diagnosis and genetic therapy. The aim of the research is to detect the KIT mutations in our patients and then analyze the relationship between KIT mutations and piebald phenotypes.23members from2families, family1includes5patients and7healthy family members, and family2includes9patients and2healthy family members. The patients were diagnosed with piebaldism clinical criteria, the specific white forelock, congenital patches of leukoderma locating on the mid-forehead, ventral chest, abdomen and extremities, the leukoderma generally keeping static in shape and distribution with typical hyperpigmentation or normal pigmentation islands within and at the borders of the depigmented areas, and the existent of positive family medical history. Then it can be made a definite diagnosis. Peripheral blood samples were collected from the families （including the patients and unaffected members） and fifty healthy controls after informed consent. Genomic DNA was extracted. Primers covering the entire coding and the exon-intron boundaries of the genes were designed, and polymerase chain reaction （PCR） was performed. PCR products were gel-purified and direct-sequenced using the ABI-PRISM3730automatic sequencer （Applied Biosystems, America）. All sequences were compared with GenBank DNA sequence （NG₀07456） to identify heterozygous mutation by using the chromas software. The identified mutation in the KIT gene was confirmed by both forward and reverse sequencing analysis. Also the clinical features and the total affected area of the body surface were collected and analyzed by using the SPSS13.0software. Two independent sample tests were carried out.Two KIT mission mutations have been identified, a recurrent mutation （c.1861G>A）, and a novel mission mutation （c.2564A>G）, both within the highly conserved tyrosine kinase domain. But the clinical phenotypes were relatively variable. Individual III-14of family1, was present with relatively mild piebaldism, with11%of the total body surface area （TBSA） affected, including congenital leukoderma on the mid forehead and depigmented patches on his knees, shins and calves. His niece, individual IV-1of the family, in contrast, presented with quite severe clinical phenotype, including patches of leukoderma on the scale and around the neck and extensive striking leukoderma of the chest, abdomen, back, arms, and legs. Five family members of family1were affected, but with very variable phenotypes, ranging from extremely severe to relatively mild. The proband of family2was present with a mild phenotype, with5%of the total body surface area （TBSA） affected, including only congenital white forelock on her mid-forehead and leukodermas on the shins and calves. All the patients detected were affected with a consistently mild piebaldism phenotype characterized by the leukoderma on the shins and calves, whilst in five cases （Ⅲ-2, Ⅲ-7, Ⅳ-1, Ⅳ-3, Ⅳ-8）, a small patch of the mid forehead leukoderma and white forelock, and in two cases （Ⅱ-1, Ⅳ-8）, limited leukoderma on the abdomen were present （Fig.1-e）. The average percentage of affected area in family2was4.44%. Statistical analysis indicated, in family1Rang1=39), Median1=21.5; while Rang2=8.5, Median2=:4in family2. There were significant differences between the two families （Z=3.003, P=0.001）. The lesion of family1was more severe than that in family2and the clinical phenotype was more variable in family1.We have identified two mission mutations within the KIT intracellular tyrosine kinase domain resulted in variable phenotypes in the research. We speculate that there must be other modifying factors, including genetic and/or environmental ones, which we have not detected, influencing the phenotypes in our family. Our further investigations will be expected to clarify the mechanisms, by which specific KIT mutations modify the gene functions and the consequent piebaldism phenotypes.The relationship between KIT mutations and piebald phenotypes is very complicated. But in China little of researchs on piebaldism have been carried out. Our research would provide new scientific theoretical basis for piebaldism prenatal diagnosis, genetic therapy and developing new ways of diagnosis and treatment. Further investigations will be expected to clarify the relationship between KIT mutations and clinical phenotypes.