The Pathogenic Sites Pseudohypoparathyroidism Pedigree Screening And The Gnas Genetic C.952 T> C Gas Function

Objective: Pseudohypoparathyroidism la (PHP la) is caused by GNAS mutation. A patient with typical clinical manifestations of PHP (round face, short stature, centripetal obesity, brachydactyly, and parathyroid hormone (PTH) resistance) presented at our center. We took further study to make sure the diagnosis and make out the influence of GNAS mutation to Gas.Methods:Further Tests were taken on the patient and her family. The Exons of the GNAS gene were amplified and sequenced with unique primers, and a novel missense mutation c.952 T>C was detected. Purchasing Kidney cDNA and Amplifying Gas cDNA with upper and lower primers carried Xhol and HindⅢenzyme site respectively. Purified PCR products were inserted into pcDNA 3.1(-) vector to yield pcDNA 3.1-GNAS-WT. The mutation in position 952 (c.952 T>C) was introduced into pcDNA 3.1-GNAS-WT using a Site-Directed Mutagenesis, products were submitted to direct sequence analysis. Plasmids were extracted using a QIAGEN Plasmid Maxi Kit. OK cells were transiently transfected with pcDNA 3.1, pcDNA 3.1-GNAS WT, and pcDNA 3.1-GNAS MT with Lipofectamine 2000. pcDNA 3.1 vector transfected cells were used as negative controls. Luminescence was measured, either treated with PTH or untreated, and normalized to the cAMP level. Same origin models were used to clarify the underlying mechanism of Gas dysfunction.Results:(1) The clinical tests showed that multi-hormone resistance only emerged in the patient; (2) GNAS sequence results showed c.952 T>C in patient and her mother; (3) Sequence results of constructed vectors showed that both vectors were successfully constructed; (4) Compared with GNAS WT, GNAS MT's function was significantly impaired, either when treated with PTH or untreated (P<0.001), while compared with control the GNAS MT can still enhance cAMP accumulation in OK cells(P＜0.01). (5) This GNAS mutation located in the conservation region, which may have some effect on the binding of Gas with Gsβγ, consequently reduce the Gas function.Conclusion:This patient was diagnosed as PHP la, which was caused by GNAS c.952 T>C mutation in this patient. An in vitro Gsa functional study showed that Gsa function was significantly impaired, this may result from Gas dysfunction in binding with Gsβγ.