Familial Hypercholesterolemia LDLR Gene CDNA Sequence Establishment And Application

【Background】Familial hypercholesterolemia (FH) is one severe single-geneautosomal-dominant genetic disease, remarkably featured by the plasma low-densitylipoprotein cholesterol (LDL-c) levels rise, the occurrence of skin and tendonsxanthoma and early onset coronary artery disease. LDLR gene mutations serve as themajor pathological basis of FH onset. So far, more than1000mutations have beenreported to lead to FM, among which two thirds are caused by point mutations andthe rest of them are due to intron mutation, frame shafts and gene deletions orfragment insertions. Currently, the popular method applied in genetic diagnosis is tosequentially sequence all the LDLR exons, consequently, with high risk to omit ormiss the synonymous mutations and intron mutations. Notably, mutations in exonsand introns may lead to deficient pre-mRNA splicing and result in15%-30%chancesfor clinical confirmed FH patients that no mutation could be detected. One study,however shed some light on the possible solution by showing that analysis on the fulllength cDNA of LDLR gene possibly to reveal the mutations that could neverdetected from the peripheral blood samples. Domestic reports have not been showndue to the limitations on mRNA acquisition and sequencing length. Therefore, thisstudy reference to foreign research results and LDLR gene binding characteristics ofChina’s population.LDLR gene cDNA sequence established method was applied topatients with FH,it tested less than mutations detected in the peripheral bloodDNA.That will provide a new method for the genetic diagnosis of FH. 【Objective】In this study, the primer sequence with reference to foreign research designoverlapping sets of LDLR gene,Ultimately select the LDLR gene optimum sevenpairs of cDNA primers. By normal LDLR gene amplification cDNA sequenceestablish the cDNA sequencing method for full length of LDLR gene.Testify thesequencing method by probing mutations that can not be detected by the traditionalsequential exon DNA sequencing method and provide new precise, reliablediagnostic method for FH patients.【Methods】1. References to foreign research design LDLR gene cDNA Sequence primers.2. Collect human peripheral blood samples from healthy subjects; apply RNAextraction kit to the collected blood samples and reversely transcribe all the RNAback into cDNA.3. Amplification the cDNA With seven pairs of the selected primers to amplifycDNA by the “Touchdown” method that developed in our lab.4. Analyze the cDNA library by bidirectional sequence alignment and piecetogether to obtain the full lengh LDLR gene.5. Confirm the preciseness of the full length DNA by comparison of the sequencewith the reference sequence in GenBank.6. Select one case with LDLR gene homozygous mutation from pool collectiondone in our lab, including50individual cases of FH patients, based on thecombined standard from “the clinical coronary heart disease” edited by ZaijiaChen,“Dutch Lipid Clinic Network Criteria”(DLCN) and “FH clinicaldiagnostic criteria in China”.7. Apply the LDLR full-length DNA sequence method to the selected case.8. Confirm the preciseness of the full length DNA by comparison of the sequence with the reference sequence in GenBank,than observed mutations.9. Collecting the human peripheral blood samples from this FH patient, apply RNAextraction kit to the collected blood samples and reversely transcribe all the RNAback into cDNA.10. Full length cDNA sequencing method was applied to the FH patient, using theabove primers amplification, matching, alignment, the patient’ mutation type, toverify the reliability of this method.【Result】1. Found unrepeatable that literature reference Tveten et designed LDLR lengthcDNA sequence primers, Combining the characteristics of China’s populationLDLR gene sequences synthesized overlapping sets of LDLR gene primers,thanaccording to the results, select the optimum seven full-length cDNA sequences ofthe primers.2. Collect human peripheral blood samples from healthy subjects; apply RNAextraction kit to the collected blood samples.Total RNA concentration was390ng/μl, A260nm/A280nm was1.91. Description mentioned very pure andwithout RNA degradation and meet the requirements of the synthesis of cDNA.3. Sequencing results showed that the seven products from amplification of cDNAof a healthy subject.4. Sequencing results showed that the seven products from amplification of cDNAof a healthy subject were all intact without any loss comparing to the GenBankdata.5. Piecing them up to make the full length of this gene and compare it with thereference sequence in GenBank revealed no difference in length.6. The testing result from Patient is in accordance with homozygous FH clinicaldiagnosis with TC of21.2mmol/L, LDL-C of19.7mmol/L, sinus rhythm on the electrocardiogram, maintenance of ST-T, normal electrocardiogram (ecg).Echocardiographic revealed the enlargement of left ventricle, mild reflux frommitral valve, no sign for calcification; Adenosine stress test and coronary bloodflow reserve remained normal. Ultrasound showed Intimal thickening occur tothe bilateral carotid artery and the initial segment of bilateral vertebral artery.7. Apply the LDLR full-length DNA sequence method to the selected case.8. The traditional diagnostic method failed to detect any pathological mutations inLDLR, apoB100and PCSK9, which led to FH.9. Collect human peripheral blood samples from FH patient; apply RNA extractionkit to the collected blood samples.Total RNA concentration was410ng/μl, A260nm/A280nm was1.89. Description mentioned very pure and without RNAdegradation and meet the requirements of the synthesis of cDNA.10. Testing results from full-length cDNA sequencing method showed that: insertionwith8bp fragment into intron8happened.【Conclusion】1. The successful synthesis of seven probes complementary to LDLR gene cDNAsequence.2. cDNA sequencing results from healthy subject showed the feasibility of thismethod.3. Traditional DNA sequencing method showed that: FH patient failed to detect anypathological mutations in LDLR, apoB100and PCSK9, which led to FH.4. Results from the full length cDNA sequencing method test showed: in Patientinsertion with8bp fragment into intron8happened, resulting in the intronsretention, which retained the splicing sites after part of the intron sequence andcausing the abnormal mRNA splicing. This abnormality led to longer RNAsplicing product and improper structural and functional proteins. Together based on previous study, the carefully designed seven probes targeting onLDLR gene cDNA was successfully applied on healthy subject and confirm thismethod. In addition, the application of the new method uncovered unidentifiedmutations, that is,8-bp in length insertion into intron8, in a FH patient who wastested by the traditional method and the result was negative for mutations. Thisresult verified this new diagnostic method and provided an improved way to identifythe mutations in FH patients with symptoms but missed by the traditional diagnosticmethod. Direct analysis according to a report in the literature in patients with fulllength cDNA sequence could detect some peripheral blood DNA mutation, so theestablishment of cDNA sequencing methods for FH provide a new method based ongene diagnosis.