A Study In The Differential Diagnosis Strategy Of Genes Associated With PSP

Purpose:Primary Spontaneous Pneumothorax (PSP) is a relatively common emergency of respiratory system, which has high recurrence rates. But its etiology remains unclear. FLCN gene mutations can result in BHDS, and the typical characteristics of BHDS are skin lesion, PSP and renal cancer. The syndrome can only appear the symptom of PSP as well. And approximately 11% of the PSP is caused by FLCN gene mutations. Recently there are two issues in the diagnosis of BHDS. Firstly, in the aspect of clinical differential diagnosis, the pulmonary symptom (such as PSP) usually appears earliest in all. How to figure out BHDS with suspected from the pulmonary clinical phenotype of PSP patients, and in order to narrow the scope of PSP patients who need to sequence the FLCN gene. Secondly, in the aspect of gene diagnosis, the type of FLCN gene mutations is quite complicated, which includes small deletions or insertions, splicing variants, stop gain SNVs and intragenic large deletions or insertions. And these variants must be detected by two different methods, such as sanger sequencing and MLPA. Even though both of the methods have high specificity, they have low-throughput and high-cost. In addition, several diseases have the symptom of pneumothorax such as MFS, LAM, EDS, Homocystinuria and alpha 1-Antitrypsin deficiency, which can be pneumothorax as first symptom. What’s worse, the atypical characteristics of these syndromes are so similar to PSP that they would be misdiagnosed as simple PSP, while other potential symptom of more serious would be ignored. Therefore, it’s necessary to sequence the genes associated with these diseases so that doctors can exclude or diagnose these atypical syndromes.Method:To solve the problem of the first, we enrolled in March 2010 to May 2014 in three hospitals of 133 cases of hospitalized patients with PSP to perform prospective experiment. The patients who have a family of pneumothorax or lung bullae in the middle or lower lobe are marked as BHDS suspected patients. Then we do Sanger sequencing and MLPA analysis in the 133 patients to verify the screening index of the specificity and sensitivity. To solve the problem of the second, we introduce a new next-generation sequencing technology called target-enrichment sequencing to do retrospective study. We screen 7 genes associated with PSP. including FLCN gene, and 30 patients of pneumothorax. All the 30 samples have sequenced for FLCN gene by Sanger sequencing and MLPA, and 20 cases of these patients are detected FLCN gene mutations including all known types, such as small deletions or insertions, splicing variants, stop gain SNVs and intragenic large deletions or insertions. And these mutations loci lie in most exons of FLCN gene. The other 7 cases without FLCN gene mutations include 2 cases (F294, F105) diagnosed as LAM,3 cases (F736, F250 and F266) suspected as BHDS,1 case (F646) diagnosed as MFS and 4 cases (F350, F602, F619 and F978) diagnosed as simple PSP. Result:Firstly,41 cases of the 133 PSP is marked as BHDS suspected patients, including 3 cases have family history, and 39 cases of the middle or lower lung bullae (including 1 case with family history).14 cases have been detected FLCN mutations by Sanger sequencing and MLPA analysis. Among them,12 cases are with middle or lower lung bullae, including 1 case has together with family history, and the other 2 cases are without middle or lower lung bullae and no family history. Finally, we calculate the sensitivity (Se) and specificity (Sp) of this method, which shows that Se is 30.8% and Sp is 97.9%. Secondly, the result of target-enrichment sequencing for FLCN gene is consistent with traditional sequencing (Sanger sequencing and MLPA). Also we discover 1 splicing variant in TSC1 gene and 1 stop gain SNV in FBN1 gene.Conclusion:Firstly, the prospective experiment has a certain degree of sensitivity and high specificity. It indicates that it’s reliable to screen BHDS suspected patients according to the pulmonary clinical symptom of PSP. Thus, we can effectively narrow the scope of PSP patients who need to sequence the FLCN gene. Secondly, The preliminary study research the application of target-enrichment sequencing in differential diagnosis of clinical syndromes associated with PSP. Then we find this method is high accuracy and it can detect all kinds of mutation types, such as small deletions or insertions, splicing variants, stop gain SNVs, intragenic large deletions or insertions and so on. While it also can be together for other PSP associated genes sequencing. So we think target-enrichment sequencing can replace the Sanger sequencing and MLPA for the differential diagnosis of clinical syndromes associated with PSP.