Multiplex ligation-dependent probe amplification of LDLR enhances molecular diagnosis of familial hypercholesterolemia

Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] typically results from mutations in the LDL receptor gene (LDLR), which are now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of genomic DNA (gDNA). However, many patients with FH have no LDLR mutation identified by this method. Part of the diagnostic gap is attributable to the genetic heterogeneity of AD FH, but another possible explanation is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. Multiplex ligation-dependent probe amplification (MLPA) is a new method that detects larger gDNA alterations that are overlooked by EBESA. We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormal LDLR MLPA pattern. In 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations, and 5 had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and then demonstrated the deleted sequence in 5 of these.These findings indicate that MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH. Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] typically results from mutations in the LDL receptor gene (LDLR), which are now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of genomic DNA (gDNA). However, many patients with FH have no LDLR mutation identified by this method. Part of the diagnostic gap is attributable to the genetic heterogeneity of AD FH, but another possible explanation is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. Multiplex ligation-dependent probe amplification (MLPA) is a new method that detects larger gDNA alterations that are overlooked by EBESA. We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormal LDLR MLPA pattern. In 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations, and 5 had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and then demonstrated the deleted sequence in 5 of these. These findings indicate that MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH. Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] affects ∼1 in 500 people and typically results from a mutation in the LDL receptor gene (LDLR; MIM 606945) (1Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolemia.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York2001: 2863-2913Google Scholar). Affected individuals have increased plasma LDL cholesterol, which without adequate diagnosis and intervention can increase the risk of fatal coronary heart disease by up to 100-fold compared with the general population (2Scientific Steering Committee on Behalf of the Simon Broome Register GroupMortality in treated heterozygous familial hypercholesterolaemia: implications for clinical management.Atherosclerosis. 1999; 142: 105-112Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Fortunately, treatment with statin drugs can substantially reduce this risk and improve clinical outcome (2Scientific Steering Committee on Behalf of the Simon Broome Register GroupMortality in treated heterozygous familial hypercholesterolaemia: implications for clinical management.Atherosclerosis. 1999; 142: 105-112Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), stressing the importance of early and accurate diagnosis. At the molecular level, FH is now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of LDLR from genomic DNA (gDNA) (3Wang J. Huff E. Janecka L. Hegele R.A. Low density lipoprotein receptor (LDLR) gene mutations in Canadian subjects with familial hypercholesterolemia, but not of French descent.Hum. Mutat. 2001; 18: 359Crossref PubMed Scopus (24) Google Scholar, 4Marks D. Thorogood M. Neil H.A.W. Humphries S. A review on the diagnosis, natural history, and treatment of familial hypercholesterolemia.Atherosclerosis. 2003; 168: 1-14Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). However, this method finds mutations in only ∼50% of clinically diagnosed FH patents (3Wang J. Huff E. Janecka L. Hegele R.A. Low density lipoprotein receptor (LDLR) gene mutations in Canadian subjects with familial hypercholesterolemia, but not of French descent.Hum. Mutat. 2001; 18: 359Crossref PubMed Scopus (24) Google Scholar, 4Marks D. Thorogood M. Neil H.A.W. Humphries S. A review on the diagnosis, natural history, and treatment of familial hypercholesterolemia.Atherosclerosis. 2003; 168: 1-14Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Part of the diagnostic gap is attributable to the heterogeneity of AD FH (5Rader D.J. Cohen J. Hobbs H.H. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J. Clin. Invest. 2003; 111: 1795-1803Crossref PubMed Scopus (475) Google Scholar). For instance, HCHOLAD2 (MIM 144010), which results from a missense mutation in APOB affecting the LDL receptor binding domain of apolipoprotein B-100 (apoB-100; MIM 107730) (6Innerarity T.L. Mahley R.W. Weisgraber K.H. Bersot T.P. Krauss R.M. Vega G.L. Grundy S.M. Friedl W. Davignon J. McCarthy B.J. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia.J. Lipid Res. 1990; 31: 1337-1350Abstract Full Text PDF PubMed Google Scholar, 7Boren J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. The molecular mechanism for the genetic disorder familial defective apolipoprotein B100.J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), accounts for 5–10% of the AD FH phenotype. A rare FH subtype called HCHOLAD3 (MIM 603776) results from mutations in PCSK9 (MIM 607786), encoding neural apoptosis-regulated convertase-1 (5Rader D.J. Cohen J. Hobbs H.H. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J. Clin. Invest. 2003; 111: 1795-1803Crossref PubMed Scopus (475) Google Scholar, 8Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Derre A. Villeger L. Farnier M. Beucler I. Bruckert E. Chambaz J. Chanu B. Lecerf J.M. Luc G. Moulin P. Weissenbach J. Prat A. Krempf M. Junien C. Seidah N.G. Boileau C. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2168) Google Scholar, 9Park S.W. Moon Y.A. Horton J.D. Post-transcriptional regulation of LDL receptor protein by proprotein convertase subtilisin/kexin type 9a (PCSK9) in mouse liver.J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). A similarly rare autosomal recessive FH subtype called HCHOLAR1 (MIM 603813) results from mutations in ARH (MIM 605747), encoding a putative adaptor for the LDL receptor (10Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.Science. 2001; 292: 1394-1398Crossref PubMed Scopus (467) Google Scholar). However, even after accounting for genetic heterogeneity, many clinically diagnosed FH patients have no LDLR mutation with EBESA. Another possible explanation for the gap in FH molecular diagnosis is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions. The larger gDNA alterations create effective hemizygosity for single exons and result in an EBESA profile that is indistinguishable from homozygosity for two normal LDLR alleles. Multiplex ligation-dependent probe amplification (MLPA) is a new analytical method (11Schouten J.P. McElgunn C.J. Waaijer R. Zwijnenburg D. Diepvens F. Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2092) Google Scholar) that detects larger gDNA deletions or insertions that would otherwise be overlooked by EBESA (12Ainsworth P. Koscinski D. Fraser B. Stuart J. Family cancer histories predictive of a high risk of hereditary non-polyposis colorectal cancer associate significantly with a genomic rearrangement in hMSH2 or hMLH1.Clin. Genet. 2004; 66: 183-188Crossref PubMed Scopus (10) Google Scholar). We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormality detectable using MLPA. Of 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations. Five had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and confirmed the deleted sequences in 5. MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH. We studied 70 unrelated Caucasian patients from southwestern Ontario who had each been referred to a tertiary lipid clinic for diagnosis and treatment of hypercholesterolemia. Each subject had a clinical diagnosis (made by one clinician) of FH according to validated clinical and biochemical criteria (7Boren J. Ekstrom U. Agren B. Nilsson-Ehle P. Innerarity T.L. The molecular mechanism for the genetic disorder familial defective apolipoprotein B100.J. Biol. Chem. 2001; 276: 9214-9218Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This study had the approval of our ethics review panel; all subjects gave informed consent. From gDNA extracted with the Puregene kit (Gentra Systems, Minneapolis, MN), EBESA of the LDLR was performed as described (4Marks D. Thorogood M. Neil H.A.W. Humphries S. A review on the diagnosis, natural history, and treatment of familial hypercholesterolemia.Atherosclerosis. 2003; 168: 1-14Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) by sequencing both strands of the promoter region and of the translated parts of all 18 exons (primer information and sequences available upon request). In addition, APOB exon 26, which harbored the receptor binding domain of apo B-100, including the recurrent R3500Q mutation in HCHOLAD2, was examined in all patients using direct sequence analysis. Standard DNA sequencing reactions using the Big Dye Terminator cycle sequencing kit version 3.1 (PE-Applied Biosystems, Mississauga, Ontario, Canada) were analyzed using a 3730 Automated DNA Sequencer (PE-Applied Biosystems) according to the protocols of the London Regional Genomics Centre (www.lrgc.ca). The SALSA P062 LDLR MLPA kit was obtained from MRC-Holland (Amsterdam, the Netherlands). The principles and stages of MLPA have been previously described (11Schouten J.P. McElgunn C.J. Waaijer R. Zwijnenburg D. Diepvens F. Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2092) Google Scholar). Briefly, each pair of diagnostic probes is designed such that the probes hybridize immediately adjacent to each other on the same gDNA target in preparation for a subsequent ligation step. Each probe consisted of a target-specific sequence and either a common forward or reverse primer-specific sequence. One probe from each pair contained a “stuffer” sequence of 19–364 nucleotides (nt), which was unique and thus diagnostic for the particular probe pair. The LDLR MLPA kit contained 31 sets of probes, 16 of which were LDLR specific and the others were control standards. Reactions were carried out in 200 μl tubes in a model 9700 thermocycler (PE-Applied Biosystems). One hundred to 300 ng of gDNA from each subject was diluted in 5 μl of 1× Tris-EDTA and denatured at 98°C for 5 min. MLPA buffer and probe mix (1.5 μl of each) were then added to allow probes to anneal to target gDNA by heating at 95°C for 1 min and incubating at 60°C for 16 h. Annealed probes were ligated at 54°C for 15 min followed by inactivation at 98°C for 5 min. Ten microliters of ligation reaction was removed for multiplex amplification using a pair of common primers, of which one was labeled with the fluorescent dye FAM. Taq polymerase was added to the reaction at 60°C, followed by 33 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and a final extension step of 72°C for 20 min. Two microliters of reaction solution was used for fragment analysis on the 3730 capillary sequencer, with LIZ-500 size standards (PE-Applied Biosystems). The procedure was performed according to the manufacturer's instructions (www.mrc-holland.com). Data analysis was performed using GeneMapper version 3.5 (PE-Applied Biosystems). Electropherograms of fragments from MLPA analysis of LDLR from normal subjects show a profile composed of 31 peaks (range, 130–382 nt). Exons 10 and 13 are not interrogated with the current version of the probe set. The multiplex contained five internal DNA quantity (DQ) control fragments. Four of these, of 64, 70, 76, and 82 nt, were ligation independent and were included to demonstrate that sufficient template DNA was present for the entire multiplex amplification reaction. The fifth DQ fragment, of 94 nt, indicated successful ligation by producing a peak of comparable size to that of the other chromosome-specific probes in the multiplex. The relative areas under the curve (AUCs) for peaks in each sample were determined. The relative peak AUC for each probe was calculated using four adjacent peaks, two on either side, as internal controls. The fraction of each peak was then divided by the median peak fraction of the corresponding fragment from five normal control samples. In 18 normal individuals, these calculations gave values close to 1.0, which corresponded to the normalized mean peak area and standard for an with two of the target sequence. results or were were performed on an were used for all the of normal with of the that a MLPA peak in this study sample corresponded to a deleted region of we identified five patients with a MLPA each ∼50% of of exon of these patients had French to that this particular MLPA have from the deletion at the of the LDLR. This deletion is the common cause of FH in French (1Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolemia.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York2001: 2863-2913Google Scholar, J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar). We used the method J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar) to sequence the deletion in these five Briefly, and were used to a fragment the deletion this fragment was and and a fragment, which indicated the of the J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar), Among the sample of 70 FH patients, were and the was The mean for and were and values for plasma LDL were and for the and of patients had and had FH patients of the were heterozygous for a of 18 LDLR missense mutations, and FH patients of the were heterozygous for a of RNA mutations, and Two FH patients of the were each heterozygous for an and FH patients of the were heterozygous for a of mutations, and Among the mutations are five LDLR mutations, which are indicated by LDLR mutations were from the of control Five FH patients of the had the APOB R3500Q mutation. FH patients of the had no LDLR mutation by EBESA and not have the APOB R3500Q mutation. The 21 FH patients who had a mutation in LDLR by EBESA the APOB R3500Q mutation were studied using LDLR MLPA. In addition, LDLR MLPA was performed on gDNA from normal LDLR deletion mutations were apparent on of MLPA peak This is in in which of peak of a probe LDLR and control peaks indicated a gene mutation. peak areas for exons were using the normal as described in Standard of of the normalized mean were for all using the of the relative of region We values for the of abnormal results at and to standard from the Among the 21 previously FH patients, were abnormal MLPA patterns in 12 Four peaks for one exon 1 and peaks for and MLPA of FH patients who had missense mutations in LDLR were all normal not peak areas in normal subjects and results from FH are by is the of the the For of normalized peak AUC for LDLR exons from MLPA from 18 normal and standard are The for FH subjects by MLPA according to the AUC was was or compared with the normalized and only areas that this are from five FH subjects MLPA had peak areas for exon a that we as The genomic DNA (gDNA) from these five subjects was MLPA from a FH subject with a peak area for exon a that we MLPA from a FH subject with a peak area for exon a that we MLPA from a FH subject with a peak area for exon a that we MLPA from FH subjects with peak areas for exons 1 to MLPA from a FH subject with peak areas for exons to MLPA from a FH subject with peak areas for exons to normalized peak in and to MLPA in and of the study sample according to molecular MLPA gene encoding apolipoprotein plasma of density lipoprotein multiplex ligation-dependent probe in a new gene encoding apolipoprotein plasma of density lipoprotein multiplex ligation-dependent probe that an abnormal MLPA corresponded to a specific gDNA sequence we a gDNA fragment that harbored the deletion is no procedure at present to to deleted gDNA the deleted region was with a method. We that the five subjects with the MLPA corresponding to 1 each had French This to that this abnormal MLPA have been the result of the deletion at the of the LDLR that is the cause of FH in patients J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar). We used the method to detect in these five patients J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar). We found that each had the diagnostic that only be the deletion was present J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar), that each was heterozygous for this fragment, and that the sequence of this fragment corresponded to J. M. G. E. The deletion French Canadian mutation in familial hypercholesterolemia: polymerase diagnostic and in Genet. 2004; PubMed Scopus Google Scholar) in all five subjects We patients the molecular for missense for for either mutations or abnormal MLPA for APOB and no LDLR mutation and normal APOB receptor binding domain sequence The mean LDL the small of 12 FH patients with abnormal LDLR MLPA patterns was not from that of the other patients by using The findings of this are as of of FH patients without the APOB R3500Q mutation had no LDLR mutation detected by EBESA of and of of FH patients with no LDLR mutation detected by EBESA had an abnormal LDLR MLPA pattern. Five of 70 FH subjects had the APOB R3500Q mutation, which is with results from other study (6Innerarity T.L. Mahley R.W. Weisgraber K.H. Bersot T.P. Krauss R.M. Vega G.L. Grundy S.M. Friedl W. Davignon J. McCarthy B.J. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia.J. Lipid Res. 1990; 31: 1337-1350Abstract Full Text PDF PubMed Google Scholar). The abnormal MLPA in five patients was confirmed by sequencing to to a specific at the of LDLR. LDLR missense mutations are common in FH, MLPA to be These results indicate that MLPA be a method to detect gDNA of the LDLR is an of the mutation type For instance, in the the LDLR mutation type in FH was gDNA such as large insertions and deletions H.H. Brown M.S. J.L. The LDL receptor in familial hypercholesterolemia: analysis of a Genet. 1990; PubMed Scopus Google Scholar). One for this was the relative LDLR of sequences that were to or exon H.H. Brown M.S. J.L. The LDL receptor in familial hypercholesterolemia: analysis of a Genet. 1990; PubMed Scopus Google Scholar). another was the that LDLR mutations in the were detected by were for gDNA of target which was the for DNA fragments. However, of small mutations was the a mutation a for a was for LDLR by the early these a large of FH mutations (1Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolemia.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York2001: 2863-2913Google Scholar, H.H. Brown M.S. J.L. The LDL receptor in familial hypercholesterolemia: analysis of a Genet. 1990; PubMed Scopus Google Scholar). to detect small mutations from gDNA using and with and of the LDLR mutations in FH that are in current are of the by exon-by-exon screening such as EBESA However, of gDNA are to of A large deletion affecting one LDLR may be detected by is no target sequence upon which amplification can the normal is effective hemizygosity for that region of of the of such a reaction in the for a DNA M. K. of of DNA by as PubMed Scopus Google Scholar) or by EBESA, a MLPA detects alterations a large gDNA region but no and is a internal standards and are for each but this is by the high by fragment for LDLR MLPA analysis including and was which the in molecular diagnosis. is no procedure for and for of abnormal for MLPA MLPA and new for of gene Mutat. 2004; PubMed Scopus Google Scholar). For clinical of LDLR MLPA to be by analysis of FH that have gDNA including and subjects with other mutation types, to be as and was that we the deletion at the of LDLR in five subjects MLPA analysis the 1 pattern. MLPA the of FH patients who had no apparent abnormality in gDNA to of is the of the FH in the remaining of these patients have had other LDLR mutation types, such as mutations or the region of the promoter and that be by our EBESA and MLPA some patients may have had another genetic cause for FH as by the five patients with APOB We used EBESA to PCSK9 (MIM and ARH (MIM in the FH patients with no LDLR mutation and normal APOB exon sequence and found no mutations in these not Mutations in PCSK9 and ARH be found in some of these FH patients MLPA. mutations in some of these FH patients may be found in other In we abnormal MLPA patterns in a of FH patients who had a LDLR mutation using EBESA the APOB R3500Q mutation. MLPA increased the LDLR mutation in FH subjects with no APOB R3500Q mutation from to 44 of to of this in mutation can be MLPA a useful to screening for the molecular diagnosis of FH. MLPA can be of such criteria as and However, the clinical importance of a FH diagnosis (2Scientific Steering Committee on Behalf of the Simon Broome Register GroupMortality in treated heterozygous familial hypercholesterolaemia: implications for clinical management.Atherosclerosis. 1999; 142: 105-112Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) the to molecular diagnostic to FH. The the of molecular heterogeneity of FH. and with analysis and This study was by a in and a from the and of from the Canadian for the and of Ontario, the Canadian the Canadian the Ontario and and the

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