Exome Sequencing Identified Causal Gene CARD9 For Corynespora Cassiicola Infection

Background We herein report one case of phaeohyphomycosis caused by Corynespora cassiicola with prominent tissue necrosis in a woman.The patient is a37-year-old female when she visited our clinic complaining of swelling and pain in her face.Two years prior,nail-sized erythematous plaques appeared on both sides of the nose.Before ten days,she was treated with glucocorticoids,prominent facial swelling with plaques rapidly extending to the forehead was noted.She was diagnosed by drug eruption or Sarcoidosis.The diagnosis of deep dermatophytosis caused by C.cassiicola was made on the basis of the clinical,histopathological,and mycological findings.This patient was not a farmer,she had no known immunosuppressive conditions,and did not respond well to systemic antifungal agents,which strongly suggested that some genetic defects concerning immune response to fungal infection should be considered thus,we carried out whole-exome sequencing on the patient.Objective To isolate and identify the pathogenic fungi,then we used the whole genome exome sequencing strategy to identify the causative gene of isolated fungi of the patient.Methods Part 1 : A 37-year-old female appeared on both sides of the nose nail-size derythematous plaques over two years.Sarcoidosis was the suggested diagnosis at that time.She denied a premorbid history of taking immunosuppressive drugs,diabetes mellitus,or local trauma involving soil or plants.On admittance,she underwent an examination showing extensive,dark red,infiltrative plaques on her face with a purulent,foul-smelling discharge,along with postauricular lymphadenopathy.Examination of other systems were unremarkable.Based on the patient's history and clinical manifestations,cutaneous tumor or infectious disease was taken into account.Microscopic examination showed many fungal spores and filaments,indicating the diagnosis of infectious fungal disease.Histologically,intense inflammatory infiltration with dematiaceous fungi could be observed.According to the morphological characteristics of the tissue cultures,Corynespora cassiicola was identified,and was later confirmed by sequence analysis of the internal transcribed spacer(ITS)regions.Analysis revealed 100%(559/559)identity to those of C.cassiicola(accession Nos.JQ595296,FJ852576 and AY238605)using the BLAST program(http://blast.ncbi.nlm.nih.gov/Blast.cgi).The diagnosis of deep dermatophytosis caused by C.cassiicola was made on the basis of the clinical,histopathological,and mycological findings.Amphotericin B treatment was initiated,and a improvement of the lesions was noted after 2 weeks' intake with a total dose of 800 mg.Part 2:(1)With written informed consent,we performed whole-exome sequencing in a Chinese patients.Genomic DNA was extracted from peripheral blood cells and whole-exome sequencing was performed.Genomic DNA was fragmented and enriched for exome sequences using Illumina Tru Seq Exome Enrichment Kit and then sequenced by using the Hi Seq 2000 platform.(2)To eliminate nonpathogenic variants,we filtered out the single nucleotide variants and short insertions and deletions(indels)existing in the public variation databases db SNP138.(3)As a result,949 different genes were detected in the patient.When we compared these variants with the genes previously reported to be implicated in fungal infection,only one gene(CARD9)was matched.(4)Sanger sequencing was subsequently performed to validate the patient's involvement with CARD9.Result(1)This case was phaeohyphomycosis caused by C.cassiicola.The patient had been treated by Amphotericin B,and a improvement of the lesions was noted after 2 weeks' intake with a total dose of 690 mg.(2)We obtained SNP and indels of one sample by whole genome exome sequencing.(3)After filtering out synonymous mutations?common mutations and the normal individual mutations that have been published in the public,we selecte the variants that were absent from most updated db SNP database for further analysis which are more likely to be pathogenic mutations.We found 949 variants in the affected individual.Only one mutation was found to be located within the CARD9 gene.(4)We analyzed the mutation in 100 controns available unaffected individuals by Sanger sequencing.We confirmed one insert mutation c.191-192 Ins TGCT,p.L64 fs X59 in the study.Conclusions(1)This study identified CARD9 gene as the disease gene for subcutaneous phaeohyphomycosis caused by Corynespora cassiicola by combining exome sequencing in a Chinese patient.(2)We had found a insert mutation(c.191-192 Ins TGCT,p.L64 fs X59)of CARD9 gene which had been reported in cases of subcutaneous phaeohyphomycosis caused by Phialophora verrucosa.This study contributes to elucidate the onset mechanism of the disease from the molecular levels.