Mutation In Ldlr Responsible For Familial Hypercholesterolemia In Two Chinese Families

Background:Familial hypercholesterolemia?FH;MIM #143890?is a common autosomal-dominant genetic disorder.The main clinical features are high levels of circulating low density lipoprotein?ldl-c?,characteristic of melanoma and develops atherosclerosis rapidly in early life.The most serious is early onset of cardiovascular atherosclerosis.FH is mainly caused by mutations in the genes coding for the LDL receptor,apolipoprotein B,and proprotein convertase subtilisin/kexin9.The three genes that cause familial hypercholesterolemia are currently recognized.FH is mostly caused by mutations within LDLR,which leads to no expression or malfunction of the protein.Low density lipoprotein receptor?LDLR?,mainly expressed on the surface of hepatic cell membrane,is responsible for the uptake of low-density lipoprotein particles?LDL?to remove cholesterol from the plasma.LDLR precursors of 120 kDa were synthesized by endoplasmic reticulum,glycosylaed in golgi body to mature type of 160 kDa,which was then transported to the cell membrane and plays a role.LDLR protein has 5 functional domains.The different domains in the low-density lipoprotein?LDL?receptor protein are encoded by specific regions in the LDL receptor gene.The first domain of the LDL receptor consists of the NH2-terminal 292 amino acids,This region of the receptor contains the site whereby apoB/apoE can binds to.The second domain of the LDL receptor is 35% homologous to a portion of the extracellular domain of the precursor for epidermal growth factor?EGF?.The region is believed to bind to PCSK9 and is associated with LDLR degradation in lysosomes.The third domain of the LDL receptor lies immediately external to the membrane-spanning domain.this region contains the clustered O-linked sugar chains.The fourth domain consists of a stretch of 22 hydrophobic amino acids that span the plasma membrane.The fifth domain is the cytoplasmic tail.It plays an important role in clustering in coated pits.The apolipoprotein B-binding domain ofthe receptor binds to circulating LDL particles,and the receptor–ligand complex isinternalized through clathrin-coated vesicles,which fuse with early endosomes.Internalization depends on the NPXY motif within the cytoplasmic tail of LDLR,which is the binding site for several proteins,including LDLR adaptor protein 1?LDLRAP1?and a multiprotein complex called CCC that includes coiled-coil domaincontaining proteins;As the LDLR–LDL complex is exposed to lower pH in the endosome,the receptor dissociates and is recycled to the cell surface.This process is called receptor circulationAccording to the nature and location of the mutations within the LDLR and to the phenotypic effects on the protein,mutations have been divided into five different classes: Class 1: no detectable LDLR synthesis;Class 2: defective LDLR transport;Class 3: impaired LDL to LDLR binding;Class 4: no LDLR/LDL internalization due to defective clustering in clathrin coated pits;and Class 5: no LDLR recycling.Proprotein convertase subtilisin kexin 9?PCSK9?is a serine protease that is secreted by the liver.It binds the LDLR expressed principally on liver cells.Once the PCSK9-LDLR complex is internalized into the hepatocyte physiologically through the coated pit region of its outer membrane,LDL receptors bound to PCSK9 are prevented from recycling to the cell surface and undergo destruction inside liver cells.The main ligand for the low density lipoprotein receptor receptor is the low density lipoprotein?LDL?.It contains a single copy of apolipoprotein B-100.About 65-70% of plasma cholesterol circulates in the form of LDL.The plasma concentration of low density lipoprotein in heterozygotes familial hypercholesterolemia?HeFH?patients is usually 5 13 mmol/L,while the level of low density lipoprotein in patients with homozygotic familial hypercholesterolemia?HoFH?is much higher than 13 mmol/L.A meta-analysis of 2,458,456 people from 19 surveys indicate that the incidence of HeFH is about 1:250 and homozygous FH is about 1:300,000?between 1:160,000 and 1:1,000,000?.Untreated HeFH tends to experience the first cardiovascular event at age 20 or earlier.More than 20 years earlier than the general population.Untreated homozygous familial hypercholesterolemia has a worse prognosis,with many patients experiencing coronary events in childhood or adolescence.High doses of statins are the first line of treatment for heterozygotic familial hypercholesterolemia.In patients with a reduced LDL level of less than 50% in the most tolerable statins,other drugs to reduce LDL are recommended.Homozygous familial hypercholesterolemia requires more professional and aggressive drug lipid-lowering therapy and even hemodialysis.Objective: LDLR gene,located on chromosome 19 p,comprises 18 exons,introns,and the promoter region.More than 1,300 LDLR mutations have been found till now.Previous studies suggested that LDLR mutation may not definite lead to FH.Therefore,it is necessary to verify the function of LDLR mutated gene.We selected two non-consanguineous FH families and sequenced their LDLR gene.After the discovery of LDLR mutation,the distribution and function of LDLR were further verified.Method: Family hypercholesterolemia families were recruited according to the diagnostic criteria of DLCNC?Dutch lipid clinic network criteria?.Family members were called to extract the fasting blood and test the blood lipids.Family tree diagram was drawed.The proband blood samples were sequenced by full exon capture,compare the genes related to cholesterol metabolism to find mutation.The mutation of LDLR gene was found in both families.For the 1st family,since the mutant site was at the last basic group of exon 16,RNA was extracted from the family members and reverse transcribed into cDNA.Primer across exon 16 was designed according to LDLR mRNA sequence,PCR amplification and gel electrophoresis,purified and sequenced.For the 2nd family,since it's not a single base change,to determine whether there is a single allele mutation or both alleles,the primers around LDLR mutation were designed,with restrictive endonuclease sequences at both ends,PCR amplification.PCR products were connected to plasmids and sent for sequencing.In order to study whether mutations affect LDLR function,Lentiviral vectors of wild-type and mutant LDLR were constructed.Virus was packaged and hepatogenic cells HepG2 were infected.Cell lines overexpressing wild-type and mutant LDLR were established.Membrane protein extracted,the content of LDLR in cell membrane and total LDLR in cells were observed by immunoblot test.The cell lines were made into slivers,stained,and the distribution of LDLR in subcellular structure was observed under confocal microscopy.In addition,LDL labeled by fluorescent substance DiI was co-incubated with cells.Fluorescence intensity of DiI-LDL in cells was analyzed by laser confocal scanning and ImageJ software.It illustrate the ability of LDLR to induce LDL uptake.Results: C.2389G>A mutation was found in proband 1 by total exon capture and sequencing.It located at the last digit of exon 16.Further analysis of mRNA found that the mutated LDLR gene cause exon 16 deletion after transcription lead to deletion of exon 16 in mature mRNA.We constructed lentivirus plasmids carrying LDLR with exon 16 deletion,packaged virus,infected HepG2 cells,detected expression and distribution of the protein.The results showed that the protein could hardly reach the cytoplasmic membrane and mainly accumulated in golgi body.For the proband 2,the mutation is located in exon 13.After PCR amplification,the product was connected to the plasmid,and the two alleles were analyzed respectively.We found that the mutation is a deletion-insertion mutation located in one allele,c.1885₁889 delins GATCATCAACC,lead 629F?phenylalanine?,630S?serine?was deleted,replaced by D?aspartic acid?H?histidine?Q?glutamine?P?proline?.This mutation results in a significant reduction in the distribution of the encoded protein in the cell membrane,and the molecular weight decreased by about 40 KD.It accumulates mainly in the endoplasmic reticulum.The HepG2 cells expressing mutant LDLR absorbed DiI-LDL significantly less than those expressing wild-type LDLR.Conclusion: We found c.2389G>A mutation and c.1885₁889 delins GATCATCAACC mutation in two hypercholesterolemia families.The former leads to abnormal mRNA splicing,deletion of exon 16 in mature mRNA,cause the membrane acrossing region of the LDLR truncated.The mutant LDLR cannot reach the cell membrane.Mediates the loss of LDL uptake function.The latter causes changes in the amino acid sequence in the LDLR EGF-homologous domain,cause LDLR to remain in the endoplasmic reticulum,the distribution on the cell membrane was significantly reduced,the ability to intake LDL goes down.Our results suggest that both mutations are pathogenic mutations of hypercholesterolemia.