Genotype And Phenotype Correlations Of 19 TTR Amyloidosis Families And Tetramer Stability Assay Of TTR^p.Ala97Ser Mutant

Objective:Transthyretin amyloidosis is a rare autosomal dominant hereditary disease caused by TTR gene mutations with TTR amyloid depositing in multiple systems.This article aims to analyze the genotype-phenotype characteristics of nineteen families with transthyretin amyloidosis in our center and to measure the serum TTR tetramer concentration,kinetic and thermodynamic stability of the hotspot mutant p.Ala97Ser(TTR^A97S),as well as the effects of TTR tetramer stabilizers on TTR depolymerization.Methods:（1）Nineteen families with Transthyretin amyloidosis（including twenty-two patients and two asymptomatic carriers）who referred to the Department of Neurology at Third Xiangya Hospital from2015 to 2022 were included.Clinical data were collected and genetic diagnosis was performed by Sanger sequencing or whole exome sequencing.The genotype and phenotype characteristics of nineteen families with transthyretin amyloidosis were analyzed.（2）The peripheral blood samples of three patients with TTR^A97Smutation,two asymptomatic carriers,and two healthy controls were collected and the serum TTR tetramer concentrations of the seven blood samples were measured.Kinetic and thermodynamic stability of synthetic wild-type TTR(TTR^WT),heterozygous and homozygous TTR^A97S were measured.Diflunisal,Tafamidis,and AG10,were added to react with homozygous and heterozygous TTR^A97S,respectively,and their effects on TTR tetramer depolymerization were measured.Results:（1）（1）Among the nineteen families with transthyretin amyloidosis,eighteen families presented with polyneuropathy（ATTR-PN）combined with possible cardiomyopathy,and one family with leptomeningeal amyloidosis.（2）The mean age at onset of twenty-two patients was 48.1±11.5 years,the mean time from onset to present of twenty-two patients was 5.4±2.4 years,the mean disease duration of the deceased patients was 6.4±2.3 years,and the mean diagnosis time of twenty-two patients was 3.5±2.3 years.（3）Neurophysiological examinations of twenty patients showed peripheral neuropathy with predominant axonal involvement.（4）Congo red staining of peripheral nerve biopsy was positive in three patients and negative in two patients.（5）Echocardiography of nineteen patients all showed ventricular septal or posterior left ventricular wall thickening（≥12mm）,five patients showed sparkling texture of myocardium,and the left ventricular ejection fractions of two patients were below 50%.（6）The brain MRI of two patients revealed extensive leptomeningeal enhancement over the surface of the brain.（2）Eleven missense mutations were identified in nineteen families with transthyretin amyloidosis,including c.112G>A（p.Asp18Asn）(TTR^D18N),c.113A>G（p.Asp18Gly）(TTR^D18G),c.128G>A（p.Ser23Asn）(TTR^S23N),c.164A>C（p.Lys35Thr）(TTR^K35T),c.165G>T（p.Lys35Asn）(TTR^K35N),c.200G>A（p.Gly47Glu）(TTR^G47E),c.214T>C（p.Ser52Pro）(TTR^S52P),c.220G>C（p.Glu54Gln）(TTR^E54Q),c.220G>A（p.Glu54Lys）(TTR^E54K),c.242A>G（p.Glu61Gly）(TTR^E61G),c.349G>T（p.Ala97Ser）.TTR^D18N,TTR^S23N and TTR^E61G were reported in mainland of China for the first time.The TTR^A97S mutation was identified in eight families,accounting for 42.1%of nineteen families in this cohort,and it was a hotspot mutant in this group.At present,the patients with TTR^A97Smutation reported in the literature were from southern of China or were ethnic Chinese.（3）The patients with TTR^A97S mutation mainly presented with symptoms of sensorimotor and cardiac involvement,the patients with TTR^D18N and TTR^S23N mutations presented with prominent symptoms of cardiac involvement.The patients of TTR^K35T mutation often presented with vitreous opacity.The patients with TTR^D18G mutations showed prominent leptomeningeal involvement.（4）（1）The TTR tetramer concentrations in serum of the patients and asymptomatic carriers with TTR^A97S mutation were lower than that in healthy controls(TTR^WT).（2）The thermodynamic stability of heterozygous TTR^A97S（C_m=3.4M）is consistent with TTR^WT（C_m=3.4M）,and the thermodynamic stability of homozygous TTR^A97S（C_m=3.2M）is lower than TTR^WT（C_m=3.4M）.（3）The kinetic stability of heterozygous TTR^A97S(t_1/2=22.2h)was lower than TTR^WT(t_1/2=42h),and kinetic stability of homozygous TTR^A97S(t_1/2=152.9h)was significantly higher than TTR^WT(t_1/2=42h).（4）All the three small-molecule TTR tetramer stabilizers（Tafamidis,diflunisal,and AG10）inhibited the formation of TTR^A97S amyloid fibrils by more than 50%when the concentrations of TTR tetramer stabilizers were higher than 7.2μM.Conclusion:（1）TTR^A97S is a hotspot mutant in southern of China and Southeast Asia,and its clinical manifestations were ATTR-PN combined with cardiomyopathy which was the most common phenotype of transthyretin amyloidosis.Patients with TTR^K35Tmutation often presented with vitreous opacity.Patients with TTR^D18G mutation showed leptomeningeal involvement.（2）The stability of serum TTR tetramer in patients with TTR^A97Smutation was lower than that in healthy controls.The depolymerization rate of heterozygous TTR^A97Swas faster than that of homozygous TTR^A97Sand TTR^WT.Diflunisal,Tafamidis and AG10 had obvious inhibitory effects on the depolymerization of TTR^A97Stetramer.