Identifying Patients With BHD Syndrome Among PSP Population From Genetic And Clinical Findings

Background:Birt-Hogg-Dube Syndrome（BHDS）caused by flcn germline mutation is a rare autosomal dominant genodermatosis that characterized fibrofolliculomas and/or trichodiscomas,pulmonary cysts（LC）,spontaneous pneumothorax（SP）,and renal tumors.A portion of patients are often misdiagnosed as primary spontaneous pneumothorax（PSP）,in that the only clinical features of whom are multiple LC and SP.These BHDS patients,named as FLCN-PSP（F-PSP）in this report,presenting neither cutaneous lesions nor renal manifestations,however are at a greater risk of developing renal cancer.Lacking the basic genetic and clinical understandings of the FLCN-PSP eventually leads to the embarrassing fact that patients with FLCN-PSP usually receive clinically misdiagnosis and mistreatmentAim:1)In this research we aim to clarify the percentage of F-PSP in PSP cohort,to study the flcn gene mutation spectrum and hot spots of patients with F-PSP,and to characterize the distinctive clinical features of FLCN-PSP that differ from the common PSP,and eventually to set up an effective and feasible clinical solution to identify patients with BHDS from PSP populations.2)The methylation level of CpG islands in flcn promoter is also in research in order to discover if any epigenetic modification is associated with non-FLCN mutant PSP.3)Finally we aim to explore if any new caused gene is involved in non-FLCN mutant PSP familiesMethod:1)In genetic analysis,the direct Sanger sequencing of all the flcn translated exons and the splice signal regions was applied to 701 PSP patients to identify the sequence mutations,coupled with the multiplex ligation-dependent probe amplification（MLPA）,Q-PCR,and long range-PCR that performed to all patients to identify large intragenic deletions/duplications.2)In epigenetic analysis pyrosequencing was performed to determinate the methylation level of CpG islands in flcn promoter region of 29 PSP patients with high flcn tissue expression,31 PSP patients with normal flcn expression,9 PSP patients with low flcn expression,and 11 normal lung tissues as control.3)In clinical data analysis,a comparative study was performed between F-PSP and Non FLCN-PSP（NF-PSP）,in which clinical features,such as family history,sex ratio,BMI,age of onset,SP episode and recurrency,and distribution of LC was statistically analyzed.4)All statistics analysis was performed on SPSS Statistics 17.0 software.Analyses of dichotomized parameters and their relationship to F-PSP or NF-PSP were done using a chi-square test.Continuously measured parameters were compared between s F-PSP and NF-PSP using the Wilcoxon rank sum test.5)Whole Exome Sequencing was performed in 3 NF-PSP families,and the data was in bioinformatics analysisResult:Totally 26 unique flcn germline mutations were identified in 77 patients with F-PSP,covering 11%of the PSP cohort and 75%of the PSP families.The 26 mutations,including 18 novel mutations,were consisted of 14 frameshift mutations in 42（54%）patients,3 in-frame deletions in 19 patients（25%）,5 nonsense mutations in 5（6%）patients and one splicesite mutation in 1（1.5%）patient.Most mutations were predicted to cause premature truncation of the translated protein folliculin.Exon 6 and 11 were the most frequent sites of mutation representing 25%of patients with F-PSP,respectively.Exon 6 TTCTTCATC repeat（c.465₄74）was the first and exclusive mutation hotspot of F-PSP,in which 18（23%）patients had a germline flcn mutation The mononucleotide tract of eight cytosines in Exon 11（c.1733-1740）was the second mutation hotspot of F-PSP,and shared by BHDS,in which another 18（23%）patients had germline flcn mutations.Three large intragenic deletions were identified in 9 PSP families and one sporadic patients,covering 1.4%of the PSP cohortThe distinctive clinical characteristics of F-PSP were consisted of:1)A high PSP family history rate:52%of patients with F-PSP had a PSP family history,comparing to the 2%in NS-PSP.2)More female patients:The sex ratio of F-PSP and NF-PSP were 64:36 and 93:7（Male:Female）,respectively.3)Early age of onset:The average age of first episode of SP in patients with F-PSP was 39yr,and about 60%of patients experienced their first Sp between 25-44 yr.The average age of first episode of SP in patients with NF-PSP was 34yr,and 50%of patients experienced their first Sp before 24 yr.4)More bilateral Sp:32%of patients with F-PSP have experienced bilateral Sp,comparing to the 16%of patients with NF-PSP.5)Lower distribution of LC on CT image:LCs in 88%of F-PSP cases were located mainly in medial/lower zone or basilar lung,and preferring bilateral lungs,comparing to the typically apical LCs in NF-PSP.The epigenetic analysis showed that the CpG islands in flcn promoter region were on an unmethylated pattern in general,and not associated with the aberrant gene expression in LC tissues of PSP.The flcn promoter region methylation might not be a common factor in pathogenesis of PSP.26%of PSP families were flcn mutation negative,showing special inheritance patterns and clinical features that quite differed from F-PSP,which implied that a new gene or new mechanism might be involved in pathogenesis of PSP.Conclusion:1)Now that flcn germline mutation is found in a relatively high percentage of clinical patients with PSP,we think it is important that F-PSP be define as a subtype of PSP.Patients with F-PSP and their relative family member with a high risk of renal cancers should be clinically diagnosed and properly treated.2)Patients with F-PSP may be clinically screening from their clinical characters,as followed:1.Family history,2.Female patients,3.Age of first episode of SP,4.Bilateral SP,5.Multiple LC located on medial or low lungs and bilateral lungs.3)A complete genetic diagnosis is consisted of DNA sequencing for flcn sequence mutations and MLPA for large intragenic deletions/duplications.4)In general the CpG islands in flcn promoter region were on an unmethylated pattern,and no evidence of the flcn promoter region methylation be involved in pathogenesis of LC was found.5)26%of PSP families were flcn mutation-negative,showing unique inheritance patterns and clinical features,which might imply that a new gene or new mechanism possibly be involved in pathogenesis of PSP.