Research On The Clinical Characteristics Of Different Molecular Types Of Pseudohypoparathyroidism

BackgroundPseudohypoparathyroidism（PHP）is caused by mutations and epimutations in GNAS locus,and characterized by possibility of resistance to multiple hormones and Albright’s hereditary osteodystrophy（AHO）.PHP can be classified into the forms 1A/C,sporadic 1B,familial 1B and PHP2.The clinical symptoms and family history are often used for clinical classification.However,several researches have found overlap between different PHP subtypes.Since different genetic mechanisms and hereditary modes are involved,molecular analysis and clinical comparison based on accurate diagnosis are important.On the other hand,bone responsiveness to serum PTH in pseudohypoparathyroidism 1B（PHPIB）is controversial.Cases with osteitis fibrosa to osteosclerosis were reported,and some also observed elevated levels of bone turnover markers.Only four serial studies were carried out but no definite conclusions were drawn.Objective1)To compare the clinical characteristics of different PHP subtypes based on molecular diagnosis;2)To study if bone tissue of PHP patients is responsive to PTH.Methods1)Comparison of PHP subtypes:blood sample and clinical data of 120 clinically diagnosed PHP patients were collected during 2000 to 2016.Combined bisulfiterestriction analysis and methylation-specific multiples ligation-dependent probe amplification（MS-MLPA）were used for detection of methylation status of GNAS differentially methylated regions（DMRs）;MS-MLPA,long range PCR and quantitative PCR（qPCR）were used for detection of STX16 copy number variation;Sanger sequencing was used for genetic mutations of GNAS、PTH1R and PRKAR1.104 out of 120 patients were found to have definite molecular changes and were included in data analysis.Demographic,clinical and laboratory features were compared between 3 PHP subtypes.2)Study of bone tissue responsiveness to PTH:（1）Bone mineral density of PHP1B:clinical data of 48 adult PHP1B patients were collected.Z scores of BMD,PTH,Ca,height and weight,treatment and disease course were included in the multiple linear regression model.Fifty five non-surgical related hypoparathyroidism patients（NS-HP）were selected to be compared with PHP1B patients.Longitudinal comparison was carried out in 10 patients.（2）Bone turnover markers（BTM）of PHP patients:serum β-CTX levels and serum ALP levels of 32 and 96 PHP1B patients were collected,respectively.Serum PTH,Ca,treatment,disease course,height and weight,treatment were included in the multiple linear regression model,Twenty-six and fifty nine NS-HP patients were selected to be compared with PHP patients.Results（1）Molecular classification:Ten PHP1A/C,21 familial PHP IB,and 73 sporadic PHP1B patients were identified.Four novel GNAS mutations were discovered,including:c.1038+1G>T,c.530+2T>C,c.880₈83de1CAAG,and c.311₃12delAAG,insT.Seventeen familial PHP IB patients were found to have STX16 3kb deletion,and 2 of them were proved to have a novo deletion encompassing STX16 exon 3-9.（2）Comparison of clinical data:the most common symptoms in this series were recurrent tetany（89.4%）and epilepsy（47.1%）.Intracranial calcification had a prevalence of 94.6%and correlated with seizures（r=0.227,p=0.029）.Cataracts occurred in 56.2%PHP patients,where there was a trend of longer disease duration in patients with cataracts（p=0.051）.Statistically significant differences（p<0.05）were observed when comparing certain clinical characteristics between PHP IB and PHP1A/C patients,including onset age（10 yo vs.7 yo）,short stature（21.3 vs.70%）,rounded face（60.6 vs.100%）,brachydactyly（25.5 vs.100%）,ectopic ossification（1.1 vs.40%）,and TSH resistance（44.6 vs.90%）,respectively.（3）Characteristics of BMD in PHP1B patients:the BMD Z-score for the LS（1.14±1.41）was higher than that for the FN（-0.20±1.00,p<0.001）and the TH（0.03±1.06,p<0.001）.PTH was a negative predictor for LS-BMD Z-score（B=-0.004,p=0.028）for sporadic PHP1B patients.Z-scores for FN-and LS-BMDs after treatment increased by 0.31±0.10 and 0.58±0.12,respectively,where the increase in LS-BMD correlated with a decrease in PTH（r=-0.72,p=0.044）.All BMD Z-scores were significantly lower in PHP1B patients than NS-HP patients for the FN,LS.and TH（-0.20±1.00 vs.1.57±1.07,1.141±1.41vs.1.96±1.32,0.03±1.06 vs.1.67±1.01,respectively,all p<0.05）.（4）Characteristics of BTMs in PHP patients:Median serum β-CTX level in PHP 1B patients was 0.60ng/ml（0.10-3.20）,and there was no statistical difference between gender and age-compared sporadic and familial PHP 1B patients.Median serum β-CTX level in adolescents was higher than that in adults（1.25 vs 0.40 ng/ml,p<0.001）;Median serum β-CTX level in adult males was higher than that in adult females（0.56 vs 0.27 ng/ml,p=0.003）.Serum β-CTX level was positively related with serum PTH level for all PHP1B,sporadic PHP1B,male PHP1B and adult PHP1B patients(B=0.001、0.002、0.002 and 0.001,all p<0.05,but no statistically significant linear correlation was found in female and adolescents.Median serum β-CTX level in adult male PHP1B patients was significantly higher than that in NS-HP patients（0.56 vs 0.16 ng/ml,p=0.004）,but no significant difference was found in female and adolescents.Median serum ALP level of PHP1A/C,sporadic PHP1B and familial PHP1B patients was 270.5IU/L,142.5IU/L and 208.5 IU/L,respectively（p=0.364）.Median serum ALP level in adolescents was higher than that in adults（244 vs 74.5IU/L,p<0.001）,but no gender difference was found.No statistically significant linear correlation was found between serum ALP level and serum PTH level in PHP1A/C and familial PHP1B patients,but positive correlation was found in sporadic PHP1B patients（B=0.191,p=0.010）.Median serum ALP level was higher in PHP1B patients than in NS-HP patients,however the difference turned out to be insignificant after stratification by gender and age.ConclusionThis study is the largest single-center series on PHP patients and summarizes the clinical and genetic features of the Chinese PHP population based on molecular analysis.The following could be concluded:1.There was substantial clinical overlap between PHP1A/C and PHP1B,so molecular analysis is recommended for all PHP patients to make more accurate treatment and genetic consultation.2.Skeletal tissue in PHP1B patients may respond to PTH,where heterogenous sensitivities to PTH may exist in different regions of bone,while no definite evidence was found to prove responsiveness in PHP1A/C patients.Therefore,it is reasonable to normalize PTH levels when treating PHP1B to avoid negative effects of PTH on bone.