Genetic Screen And Clinical Characteristics Of Primaty Hyperparathyroidism Patients: A Large Sample Single-Centre Retrospective Study

BackgroundPrimary hyperparathyroidism(PHPT)is characterized by excessive secretion of parathyroid hormone(PTH)and is a leading cause of metabolic bone disease with hypercalcaemia and hypophosphataemia.The majority of cases are sporadic PHPT(SHPT),and less than 10%of cases are hereditary or part of familial syndromes.At present,the genetic screening of PHPT patients is mostly based on the Sanger sequencing,and can only screen for a handful of genes on age-specific and syndrome-specific patients,which might lead to selection bias and omit patients with atypical clinical.Till now,there is no clear data on the proportion and composition of hereditary PHPT in the Chinese population.In addition,glial cell missing 2(GCM2)has been associated with the pathogenesis of PHPT in recent years,and the relevant data in Chinese is still lacking.ObjectiveIn this study,high-throughput DNA sequencing was performed for the candidate gene screening in newly diagnosed PHPT patients of our centre:1.To clarify the proportion and composition of hereditary/familial PHPT in the Chinese population,and analyze the genotype-phenotype characteristics.2.To screen the germline variants of GCM2 in Chinese PHPT patients and to summarize the clinical features of PHPT patients associated with GCM2 mutations.3.To clarify the genetic background and clinical characteristics of MEN1 related PHPT(MHPT)patients in our centre.Methods1.Newly diagnosed PHPT Han patients who visited the endocrinology department of Peking Union Medical College Hospital(PUMCH)consecutively from August 2015 to August 2019 were collected.Those patients who consented to genetic screen were enrolled.Targeted next-generation sequencing(T-NGS,panel containing 8 PHPT related genes:MEN1,RET,CDKN1B,CaSR,HRPT2/CDC73,GNA11,AP2S1,GCM2)combined with MEN1-MLPA and CDC73-MLPA were used to screen subjects for candidate gene variants.Pathogenicity of variants was classified according to the guidelines of the American College of Medical Genetics and Genomics(ACMG).The diagnosis of hereditary/familial PHPT was based on clinical manifestations,family history and genetic screening.The clinical characteristics of hereditary PHPT and non-hereditary PHPT were compared.GCM2 variants were detected and verified in a dual-luciferase reporter gene assay.And then summarize the clinical features of PHPT patients carrying activating GCM2 variants.2.MHPT patients who visited the endocrinology department of PUMCH from January 1984 to January 2020 were collected.Those patients who consented to genetic screen were enrolled.T-NGS or Sanger sequencing combined with MEN1-MLPA were used to screen subjects for candidate gene variants.The clinical characteristics of MHPT and sporadic PHPT in the same period were compared.3.The clinical data were collected retrospectively,including general information,clinical manifestations and biochemical indexes related to PHPT,clinical evaluation of hereditary PHPT,imaging examination and surgical pathology.Bone mineral density(BMD)was measured by dual-energy x-ray absorptiometry(DXA,GE-lunar),and trabecular bone score(TBS)was calculated by the same version of TBS software(iNsight v2.1).ResultsA total of 394 consecutive PHPT patients of Han nationality were enrolled in the first and second part of the study;120 MHPT patients and 360 clinically sporadic PHPT patients diagnosed in the same period were enrolled in the third part of the study.1.Genetic screen and clinical characteristics of large sample PHPT patients in a single centre.1)Genetic variant spectrum in PHPT patientsA total of 103 patients were found to carry 79 rare non-synonymous variants,and 37 pathogenic or likely pathogenic variants were detected in 41 patients,with a variant detection rate of 10.4%.With three long-range deletions screened by MLPA,the variant detection rate was increased to 11.2%(44/394).In addition,19 of 30 clinical diagnosed MEN1 patients carried MEN1-VUS variants.With the combination of genetic screening and clinical screening,the rate of hereditary PHPT accounted for 18.8%(74/394)of the total PHPT patients in this study.2)Comparison of clinical characteristics between hereditary PHPT and non-hereditary PHPTThere were 74 patients in the hereditary group and 262 patients in the non-hereditary group(All hereditary PHPT patients and rare variants carriers were excluded).Both of the diagnosis age and onset age of patients in the hereditary group was significantly lower than that in the non-hereditary group(diagnosis age:43.6±14.0 vs.53.7±14.9,onset age:35.4±13.8 vs.50.6±14.8,P<0.001),with a higher proportion of males(44.6%vs.24.0%,P<0.001).The levels of ionized calcium and serum ?-CTX were higher in the hereditary group,while there were no significant differences in serum total calcium,PTH and other indicators.The proportion of parathyroid hyperplasia(PH)and multiple-gland involvement in the hereditary group was significantly higher than those in the non-hereditary group.3)Comparison of clinical characteristics between the variant group and non-variant groupThere were 63 patients in the variant group,including 44 patients with pathogenic/likely pathogenic variant(including 3 patients with long-range deletions),and 19 patients with MEN1-VUS mutation.There were 269 patients in the non-variant group(All patients with rare variants were excluded).The differences between the two groups were similar to those in the hereditary group and non-hereditary group,except for the proportion of carcinoma in the variant group was significantly higher than that in the non-variant group.4)Analysis of the related factors of hereditary PHPTBinary logistic regression analysis was used to analyze the key elements of the hereditary PHPT diagnosis.The results showed that when other variables remained unchanged,the odds ratio of patients with multiple-gland involvement was 6.175 times higher than that of patients with single-gland involvement;and the odds ratio was 0.967 times when the age increased by one year.According to the linear regression model,the risk probability of hereditary PHPT is higher in patients younger than 35 years old.And according to ROC curve,the sensitivity was more than 80.53%when the onset age was less than 38 years old,and the specificity was more than 81.08%when it was more than 49 years old.2.Functional verification and clinical characteristics of PHPT patients with GCM2 germline variants.Five of 394 patients carried four rare missense variants of GCM2:c.488A>G(p.E163G),c.1162A>G(p.K388E,n=2),c.1144G>A(p.V382M),and c.1247A>G(p.Y416C).The results of the Dual-Luciferase reporter gene assays suggested that two mutations(p.k388e and p.v382m)located in highly conserved regions(CCID:ss 379-395)were related to the activation of GCM2 transcription function.The mean diagnosis age of 5 patients(male/female:4/1)with GCM2 missense variation was 52.0±15.6 years old,the median serum calcium level was 3.04(3.00,3.30)mmol/1,and the median PTH level was 427.0(250.3647.0)pg/ml.All patients were treated with parathyroid exploration and adenoidectomy.Two patients with p.K388E and one patient with p.Y416C were diagnosed as PC,and the patients with the p.V382M variant was APA.One patient failed to achieve remission and refused reoperation.One patient relapsed two years after the operation and achieved remission after reoperation.The other three patients achieved remission after the first operation and did not relapse,with a follow-up time of 4.5 years,3 years and 4 years,respectively.3.MEN1 genetic screening and clinical characteristics of MHPT patients1)Screening of MEN1 germline variation in MHPT patients120 MHPT patients were included in the study,of which 103 patients carried 78 kinds of MEN1 gene variants,with a variant detection rate of 85.8%.According to ACMG,51 patients carried pathogenic/likely pathogenic variants(including 3 patients had long-range deletions of MEN1),52 patients carried VUS variants,one patient carried MEN1 synonymous variant,five patients carried VUS variants of other genes.Frameshift and missense variant accounted for the largest proportion.No hot spot variation was observed.17 patients were negative in genetic screening,with an older mean age(47.8±14.8 years)than those patients with MEN1 rare non-synonymous variant.2)Comparison of clinical characteristics between MHPT patients and SHPT patients in the same periodThere were 120 MHPT patients and 360 SHPT patients in the same period.The MHPT group consisted of 50 males and 70 females with the median age of 43.5(31.5,52.0)years,which is younger than that of SHPT patients(P<0.001).The proportion of male in MHPT group was higher.The most common clinical manifestation of MHPT patients was renal calculi/renal calcification(65 cases,54.2%),which was higher than that in the SHPT group(35.6%,P<0.001).There was no significant difference in gastrointestinal involvement,skeletal involvement,serum total calcium,ionized calcium and PTH between the two groups.In MHPT group,96 patients(80.0%)underwent surgery.43 cases had pathology of hyperplasia(44.8%),49 cases had pathology of adenoma(51.0%),three cases had pathology of atypical adenoma and one case had pathology of carcinoma(4.2%).13 patients(13.5%)did not relapse after the operation.Among 83 patients(86.5%)with remission,24 patients(24/83,28.9%)had recurrence from 0.5 to 15 years after operation.3)Skeletal involvement in MHPT patients compared to age-and sex-matchedSHPT patients A total of 86 patients with MHPT have completed BMD and TBS examinations inour hospital,and 86 SHPT patients with the same age,gender and diagnosis period were randomly matched.The bone mass in lumbar spine and TBS of MHPT patients were significantly lower than those of SHPT patients(bone mass:0.91±0.18 g/cm2 vs.1.01±0.17 g/cm2;TBS:1.22 ± 0.14 vs.1.29 ± 0.11,P<0.001).while there was no significant difference in bone mass of total hip and femoral neck between the two groups.According to TBS,among 34 MHPT patients with normal BMD,15 patients(44.1%)had bone microstructure damage and six patients(17.6%)had partial bone microstructure damage;while among 45 SHPT patients with normal BMD,6 patients(13.3%)had bone microstructure damage,and 6 patients(13.3%)had partial bone microstructure damage.ConclusionIn this study,the proportion of hereditary PHPT was determined to be 18.8%in the large-sample-sized and consecutively enrolled Chinese PHPT cohort.62 cases of hereditary PHPT(15.7%)were clinically diagnosed,and 12 cases(3.0%)were found to carry pathogenic/likely pathogenic variants through T-NGS in patients without clinical manifestations of hereditary PHPT.Positive family history,the onset age less than 40 years old,and multi-gland involvement are related factors of hereditary PHPT.It is recommended that patients with hereditary PHPT related factors should undergo clinical screening of syndrome PHPT as well as T-NGS genetic screening,and if necessary,MLPA screening may also be performed.The gain-of-function variants of GCM2 in this study are all located in CCID.The frequency of GCM2 activating variants in PHPT patients of this study is 0.8%,and the pathological type supported a higher malignant trend in cases with non-synonymous GCM2 variants.The variant detection rate of MEN1 in this study was 85.8%,and no hotspot variant were observed.Patients with negative genetic screening were older,but without significant differences in other clinical features from those with positive genetic screening.Compared with SHPT patients,MHPT patients are younger,with a higher proportion of urinary involvement,and the microstructure of cancellous bone suggested by TBS is more severely damaged.However,there is no significant difference in serum calcium and PTH between the two groups.Parathyroid hyperplasia and multi-gland involvement were more commonly seen in MHPT patients.