Clinical And Basic Study On The Regulation Of Cholesterol Metabolism By LDLR

Part I Analysis of a compound heterozygous familial hypercholesterolemia pedigree and exhaustive review of Chinese homozygous familial hypercholesterolemia patientsObjective: We presented a pedigree of a 10-year-old Chinese Ho FH boy with biallelic LDLR mutations and exhaustively reviewed the Chinese Ho FH cases to raise concern about FH in China.Methods: The proband was clinically diagnosed as Ho FH in the endocrinology department of the First Affiliated Hospital of Nanjing Medical University in 2014 and followed up for nearly 3 years.Detailed family history and clinical and biochemical data were gathered from the pedigree.Genomic DNA was isolated and the reported LDL-related genes(LDLR,APOB,PCSK9,ABCG5,ABCG8,ANGPTL3,APOC3,and LDLRAP1)were sequenced.We further performed singlenucleotide polymorphism genotyping of some other FH-unrelated genes and short tandem repeats to exclude nonpaternity.We searched for the Chinese Ho FH reports in Pubmed,CNKI,Wan Fang and other databases to exhaustively analyze the characteristics of the Chinese Ho FH patients.Results: The proband presented with extensive cutaneous and tendon xanthomas and was diagonsed as Ho FH,while his mother and grandfather were both He FH patients.The proband carried compound heterozygous LDLR disease-causing mutations,including p.(His583Tyr)variant transmitted from the mother and a de novo p.(Gln242*)variant on the paternal allele.Genotyping revealed a 99.99% likelihood of paternity.The maximum atorvastatin monotherapy(40mg daily)for over a year reduced LDL-C level from 14.87 mmol/L to 9 mmol/L(~40%)while a combination of simvastatin 40 mg daily and ezetimibe 10 mg daily resulted in another 20% lowering of LDL-C level.Furthermore,our exhaustive review of English and Chinese literature reveals that 116 Ho FH cases with detailed clinical information have been previously reported from China so far.However,genotypes were reported for only 74 of them.Conclusion: We advocate the application of genetic testing in Ho FH diagnosis and emphasize the importance of aggressive combined therapy.Based on the high prevalence of He FH,we should work together to establish an early screening management network for FH patients in China.Part II Study on the mechanism of TSH regulating Cholesterol metabolism by PCSK9Objective: TSH is closely related to abnormal lipid metabolism.PCSK9 degrades liver LDLR and plays an important role in regulating cholesterol metabolism.The purpose of this study is to explore the role of TSH in cholesterol metabolism by regulating PCSK9.Methods: We used in vitro experiments to explore signaling pathways involved in TSH upregulating PCSK9 by Western Blot,Real-time PCR,flow cytometry,small interfering RNA technology.We further established Subclinical hypothyroidism rat model to explore exogenous TSH effect on lipid and PCSK9 metabolismResults: In Hep G2 cells,LDLR expression on the plasma membrane was decreased,PCSK9 m RNA and protein levels were synchronously upregulated after rh TSH treatment,while the effects could be blocked by interfering TSH receptor(TSHR).SREBP1 C and SREBP2 m RNA expressions were enhanced after rh TSH treatment,and specific Si RNAs significantly inhibited the effects of rh TSH.Furthermore,there was a noticeable induction of PCSK9 expression by rh TSH even though HMGCR gene expression was silenced.In thyroidectomized rats,the production of endogenous thyroid hormone was eliminated and endogenous TSH with constant administration of exogenous L-T4,serum PCSK9 and TC,hepatic PCSK9 expression was increased byadministration of exogenous TSH.Conclusion: TSH could up-regulate hepatic PCSK9 expression by SREBP1 C,SREBP2,which indicated a potential mechanism for hypercholesterolemia involving direct action of TSH on the liver.