Genetic Mutation Profiles And Clinical Phenotypes In Patients With Childhood-onset Non-surgical Hypoparathyroidism

BackgroundHypoparathyroidism(HP)is a disorder of calcium and phosphorus metabolism due to parathyroid hormone(PTH)deficiency,caused by neck surgery,trauma,radiation,autoimmunity and genetic factors,etc.More than a dozen genes have been found to be related to the pathogenesis of HP so far.Studies of large-sample sized comprehensive genetic screening of HP are limited,and clinical and molecular genetic profiles of the hereditary HP are lacking.With the development of molecular biological diagnostic techniques,targeted next-generation sequencing(T-NGS)has become an economic and highly efficient method for monogenic disorders caused by mutations of multiple candidate genes.Multiplex ligation-dependent probe amplification(MLPA)is relatively simple to detect large deletions with mature commercial kits.ObjectiveIn this single-center study,large-sample sized non-surgical childhood-onset patients with HP were enrolled1)to explore the role of 15 known candidate genes in pathogenesis of hypoparathyroidism by combination of T-NGS and MLPA technology;2)to verify the pathogenicity of novel mutations in TBX1 and CaSR gene by in vitro functional experiments;3)to emphatically analyze the clinical characteristics of several types of hereditary HP based on accurate molecular diagnosis.A simplified genetic diagnosis procedure for non-surgical HP was explored.MethodsIn this study,clinical data and biochemical indicators were collected from HP patients with onset age prior to 18 years old who visited the endocrinology department of Peking Union Medical college hospital consecutively from 1975 to 2018.Secondary factors causing HP including neck surgery,trauma and radiation were excluded.Combination of the self-designed T-NGS panel containing 15 HP-related genes with TBX1-MLPA was used to screen subjects for candidate gene mutations.ACMG was used to classify the detected variants into pathogenic,likely pathogenic,variants of unknown significance(VUS),likely benign and benign.The pathogenicity of newly discovered missense mutations in TBX1 and CASR was verified by the in vitro dual luciferase reporting system.Based on the results of genetic screening and in vitro functional experiments,the clinical characteristics of hereditary HP with definite genetic diagnosis were analyzed and summarized.Patients with benign variants and without any rare variants were defined as idiopathic HP(IHP)as controls for comparision of clinical characteristics.ResultsA total of 173 non-surgical childhood-onset patients with HP were enrolled.1)Genetic mutation spectrum in non-surgical childhood-onset patients with HP By T-NGS sequencing,27 pathogenic or likely pathogenic variants were detected in 23 patients,involving 5 genes of TBX1,CASR,AIRE,GA TA3 and FAM111 A,of which 13 were newly discovered.The haploinsufficiency of TBX1 was detected in 25 patients with TBX1-MLPA.The mutation detection rate was 27.7%,and they were TBX1(15.0%),AIRE(5.2%),CASR(4.6%),GA TA 3(2.3%)and FAM111A(0.6%),respectively.In addition,12 VUS variants were detected in 11 patients,involving 5 genes of GA TA3,CASR,FAM111 A,GCM2 and PTH.2)Comparison of clinical characteristics between patients with and without mutationsBy T-NGS and TBX1-MLPA,pathogenic variants were detected in 48 patients(mutation group),and benign mutations or no mutations in 114 patients(no mutation group).In mutation group,60.4%patients displayed HP prior to 5 years,while only 7.0%in no mutation group.In all patients with onset age of HP prior to 5 years,the mutation detection rate was above 60%.Compared with no mutation group,the onset age of HP was earlier(2.8[0.1,9.6]years vs 13.0[8.8,15.0]years,P=0.000),and percentages of extra-parathyroid manifestations and positive family history were higher(P<0.05)in mutation group.3)Functional verification of some variants detected in this study and analysis of clinical characteristics in hereditary HP(1)In vitro functional verification of TBX1 mutation and clinical characteristics in DiGeorge syndrome type 1(DGS1):Among 26 patients with DGS1,25 carried the TBX1 haploinsufficiency and 1 was discovered novel TBX1 missense mutation:c.A1469G(p.Y490C).In vitro functional experiments showed that the transcriptional activity of p.Y490C mutation of TBX1 reduced by about 50%comparted with the wild type.Comparted with IHP patients,patients with DGS1 had an earlier onset age of hypocalcemia(4.5[0.2,10.3]years vs 13.0[8.8,1 5.0]years,P=0.000),mild degree of low levels of serum parathyroid hormone(6.8[3.0,15.6]pg/mL vs 3.0[3.0,3.3]pg/mL,P=0.002)in the situation of similar disease course.Moreover,DGS1 patients took lower doses of vitamin D preparation(plain vitamin D:3.25±1.62×10,000 IU/day vs 4.90 ± 1.85x1 0,000 IU/day,P=0.016;calcitriol:0.35±0.15μg/day vs 0.64 ± 0.31 μg/day,P=0.059)compared to IHP cases.In the 23 patients without congenital heart disease,despite the presence of extra-parathyroid manifestations(such as dysmorphic facies,intellectual disability,slurred speech and recurrent infections),clinical diagnosis was still difficult prior to genetic diagnosis.(2)In vitro functional verification of CASR mutation and clinical characteristics in autosomal dominant hypocalcemia type 1(ADH1):10 CASR missense mutations were detected in 11 patients,including 7 likely pathogenic variants(p.Q117R、p.D217Y、p.E241K、p.E767Q、p.F798V、p.F798C、p.Y839C)and 3 VUS variants(p.H429Q、p.A152V and p.I139T).In vitro functional experiments showed increased transcriptional activity at extracellular calcium concentrations of 0.5mM,1.0 mM,2.0 mM or 4.0 mM of the 10 variants of CaSR,with left-shifted dose-efficiency curve compared to the wild type.Therefore,in vitro functional experiemts,on the basis of T-NGS and TBX1-MLPA,expanded the overall mutation detection rate to 29.5%(51/173).The 11 patients with ADH1 had an early onset of hypocalcemia,with a median age of 0.09[0.02,1.01]years.Levels of serum PTH in 10 patients werer below normal reference range.Nine patients developed hypercalciuria after receiving calcium and vitamin D agents therapy.Seven of them had hypomagnesemia.In addition,compared with IHP patients,the onset of HP was earlier,the urinary calcium level was higher,and the occurrence rate of urinary calculi was higher in ADH1 patients(P<0.001)(3)Clinical characteristics in autoimmune polyglandular syndrome type 1(APS1):A total of 9 patients with APS1 detected in this study,during a follow-up of 21.9 ±7.9 years,showed a median of 4[1,5]components(range 1 to 7).Two of them presented typical triad manifestations,including HP,candidiasis and Addison’s disease,while 3 of them presented two of the triad.Six patients had extra-parathyroid features,including ectodermal dysplasia,hair loss,hypothyroidism,premature ovarian failure,retinitis pigmentation,and chronic diarrhea etc.(4)Clinical characteristics in other hereditary HP:Among the 4 cases with hypoparathyroidism-deafness-renal dysplasia(HDR)syndrome,3 had typical manifestations,including HP,hearing loss or renal dysplasia.Another patient was diagnosed as Kenny-Caffey syndrome type 2(KCS2)due to the manifestations of short stature,delayed closure of anterior fontanelle,cortical thickening,medullary stenosis of tubular bones,small cornea and small eyeball.ConclusionIn this large-sample sized non-surgical childhood-onset patients with HP in China,the mutation detection rate was about 30%in 15 known candidate genes.The combination of T-NGS and TBX1-MLPA technology is an effective,economic and quick method for genetic screening.In vitro function experiments are important to verify the pathogenicity of mutations.Among hereditary HP detected in this study,DGS1 is most common,and TBX1 haploinsufficiency is the most common mutation type,which can be effectively detected by TBX1-MLPA.The diagnosis rate of DGS1 is relatively low due to its varied clinical manifestation.The doses for treatment of hypocalcemia tend to be lower in DGS1 cases,suggesting a residual function of parathyroid gland.Other hereditary HP diagnosed in this study include ADH1,APS1,HDR syndrome and KCS2.The novel mutations enlarge the gene spectrum of HP.After excluding secondary factors of HP,for patients with an early onset age(especially prior to 5 years old),multiple extra-parathyroid manifestations and positive family history,TBX1-MLPA was firstly suggested due to the high incidence of DGS1.T-NGS can be considered in patients with negative result of TBX1-MLPA.