Molecular Diagnosis Of Neurofibromatosis By Multigene Panel Testing

Background Neurofibromatosis(NF)is characterized by abnormal development of the nervous system,bones,and skin.It is mainly divided into three clinical types:neurofibromatosis type I,neurofibromatosis type II and schwannomatosis,which are caused by mutations in NF1,NF2 and SMARCB1 genes,respectively.The most common form is NF1(96%),followed by NF2(3%)and the lesser known form schwannomatosis,which greatly affect the physical and mental health of patients.However,the manifestations of various types of neurofibromatosis are complex,and it is difficult to make an early and accurate diagnosis of it only by relying on clinical manifestations and routine laboratory examinations.Next-generation sequencing(NGS)is a high-output sequencing method that can rapidly sequence exomes,transcriptomes,and genomes.NGS,especially multigene panel testing,has promoted rapid progress,allowing for the simultaneous analysis of many genes and improvements in the detection rate of mutations and providing a good auxiliary diagnostic tool for the rapid clinical diagnosis of neurofibromatosis.Objective The multigene panel combined with sanger sequencing was used to confirm the diagnosis of two clinically suspected neurofibromatosis,clarify the causative gene,and provide a reliable auxiliary diagnostic tool for the clinical diagnosis and classification of neurofibromatosis.Methods First Collect clinical data and samples of two patients and their relatives.The main manifestation of patient 1 was skin tumor;histopathology showed large and isolated tumor nodules,and the spindle cells were arranged in a wave-like pattern.The immunohistochemical results showed that CD34 was diffuse and strongly positive,and S100 was scattered and weakly positive.Patient 2 had skin plaques and weak right eye as the main manifestations.Head MRI showed neurogenic tumors in the brain.Histopathology showed multiple tumor nodules of different sizes,and the spindle cells were arranged in a vortex-like arrangement.Immunohistochemical results It showed that CD34 was scattered weakly positive,and S100 was diffusely strong positive.The collected samples include peripheral blood,hair follicles,oral mucosa and skin tumors.Extract and purify sample DNA;multigene panel sequencing of DNA(Illumina Hiseq X Ten sequencing platform),use Illumina CASAVA1.8.2 software to analyze the data,and obtain mutation sites.Sanger sequencing(ABI3730XL platform)was used to verify the mutation sites;ultradeep sequencing(Illumina Hiseq X Ten sequencing platform)was used to analyze the mosaic rate of gene mutations in each tissue.Results Patient 1 main complained of cutaneous neurofibromatosis.The histopathological and immunohistochemical results were consistent with the changes of type I neurofibromatosis.Although the patient’s clinical manifestations were obvious,no mutation were found in his peripheral blood by multigene panel testing.Then a heterozygous mutation of type 1 neurofibromatosis gene was found by using multigene panel testing again.The mutation is located in exon 5 of the neurofibromatosis type I gene,c.495＿498del:p.Thr165 fs.Sanger sequencing did not find this mutation in peripheral blood,oral mucosa and hair.Although this mutation has been reported in the academic world,we have reported the somatic mosaic form for the first time.We found that patient 1 had the highest mutation rate in cutaneous neurofibromatosis in the above samples by ultradeep sequencing,and the diagnosed was mosaic type I neurofibromatosis.Patient 2 mainly presented with skin plaques,amblyopia in her right eye,and an intracranial neurogenic tumor by MRI.Histopathological and immunohistochemical results are consistent with the changes of type II neurofibromatosis.We found a heterozygous mutation type II neurofibromatosis gene in its peripheral blood by multigene panel testing combined with sanger sequencing.The mutation was located in exon 1 of the type II neurofibromatosis gene,c.36＿39del:p.Ser12 fs.This mutation was belonged to a germline mutation and had not been reported in the Chinese population.The diagnosed of patient 2 should be neurofibromatosis type II.Conclusion This study used multigene panel combined with sanger sequencing,and ultradeep sequencing methods to confirm the diagnosis and classification of neurofibromatosis in two clinically suspected neurofibromatosis patients.Although two mutation sites have been reported in this study,our findings have enriched the manifestations of neurofibromatosis gene mutations.The clinical manifestations of neurofibromatosis are complex.The application of multigene panel combined with sanger sequencing technology greatly improves the detection rate of gene mutation sites,and provides a reliable aid for the discovery of difficult gene diseases and new gene mutations.