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

Abstract

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-byexon 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 . 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 (2).Fortunately, treatment with statin drugs can substantially reduce this risk and improve clinical outcome (2), 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-byexon sequence analysis (EBESA) of LDLR from genomic DNA (gDNA) (3, 4). However, this method finds mutations in only ‫ف‬ 50% of clinically diagnosed FH patents (3, 4). Part of the diagnostic gap is attributable to the heterogeneity of AD FH (5). 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) (6, 7), 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 (5, 8, 9). 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 (10). 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 (11) that detects larger gDNA deletions or insertions that would otherwise be overlooked by EBESA (12). 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 splic-

A double heterozygote for familial hypercholesterolaemia and familial defective apolipoprotein B-100

“…The probands were reported to have an unusual phenotype of aggravated hypercholesterolaemia that was complicated with premature coronary arterial disease, although remaining responsive to The case we present in this paper is a double heterozygote for the p.Leu479Pro mutation in the LDLR gene and the common p.Arg3527Gln mutation in the APOB gene. The p.Leu479Pro mutation has been reported previously, 5,14,15 and we have found this mutation in 4/414 ( 1%) of our unrelated patients with detected mutations. Although no functional studies have been performed to confirm its pathogenicity, the p.Leu479Pro mutation occurs in the epidermal growth factor spacer domain of the LDLR protein that is required for acid-dependent dissociation of the receptor from its ligand during recycling, and the mutation may interfere with receptor recycling.…”

Update of the Portuguese Familial Hypercholesterolaemia Study

“…One of the large deletions was found in one family with a severe phenotype ( Supplementary Fig. S2); a 5-year-old girl and her father with premature CHD carried the same allele with a deletion of the 3 -end of the LDLR gene comprising exons [16][17][18]. The other large deletion, of exons 2 and 3, was found in a child, his brother and father with a severe phenotype and a family history of premature CHD.…”

Copy Number Variation in the Human Genome and Its Implications for Cardiovascular Disease

“…For instance, we recently showed that testing for both SNPs and CNVs expands the molecular diagnosis of familial hypercholesterolemia. 39 Like SNPs, smaller CNVs will affect only single genes and thus contribute, together with SNPs, to single-gene disorders. However, unlike SNPs, larger CNVs can affect 2 or more contiguous genes and thus contribute to syndromic or complex disorders caused by defects in multiple genes.…”