Analysis Of Pseudohypoparathyroidism And GNAS1 Gene

Background and ObjectivePseudohypoparathyroidism (PHP) is mainly presented as target organs resistance to parathyroid hormone (PTH), characterizing as tetany, hypocalcemia, hyperphospheremia, hypocalcinuria, low urinary phosphorus, high serum PTH. Some patients have the special physical features, such as round face, brevicollis, short stature,obesity, brachydactyly and mental retardation.PHP can be divided into several types:PHPâ… a,PHPâ… b,PHPâ… c and PHPâ…¡. The studies from foreign countries showed that PHPâ… a is caused by heterozygous inactivating GNAS1 mutations within exons 1-13 and PHPâ… b was associated with abnormal methylation of the differentially methylated Regions (DMRs) in the GNAS1 gene. GNAS1 is a complex imprinted gene that is located at human chromosome 20q13, composing of 13 exons and 12 introns, spanning about 20kb.It encodes multiple products by use of four alternative first exons and the common 2-13 exons.The mainly product is Gsa, translated from the most downstream promoter (exon 1) of GNAS1.The gene contains three differentially methylated regions (from upstream to downstream):the paternally methylated NESP55 promoter region, the maternally methylated XLa promoter region and the maternally methylated exon 1A promoter region.This study was mainly to explore whether there are mutations within the 13 exons or abnormal methylation of DMRs in the GNAS1 gene in 10 Chinese PHP patients, discussing its molecular genetics mechanism.Subjects and MethodTen PHP patients have been diagnosed and treated in our department in the past.Genomic DNA was collected from the blood samples of these patients and their parents.1-13 exons were detected by PCR and direct sequencing.The methylation status of GNAS1 gene was detected by methylation-specific PCR technique (MSP) and direct sequencing analysis. Fifty healthy people were also used as normal controls.Result1. Neither mutations of the thirteen exons nor abnormal methylation of DMRs in the GNAS1 gene was detected in three PHPâ… a patients;2. Objective bands of 1A region were amplified only by unmethylated primers in seven PHPâ… b patients, but by two pairs of primers in their parents and healthy people.All of these were confirmed by direct sequencing. There was abnormal methylation of 1A region (imprinting defect) in PHP I b patients.Target strips of NESP55 and XLa regions can be amplified by both the primers in all subjects that include seven PHP I b patients, their parents and healthy people. These results were also confirmed by direct sequencing.There were no aberrant methylation in these two regions (normal imprinting).Conclusion1. PHP patients with AHO not always have GNAS1 genetic mutation;2. Loss of exon 1A methylation (imprinting defect) is the cause of Chinese PHP I b patients;3. Application of MSP to detect GNAS1 exon 1A methylation can be used as a gene diagnostic method of the patients with PHPâ… b.