Elsevier

Journal of Clinical Lipidology

Volume 10, Issue 4, July–August 2016, Pages 944-952.e1
Journal of Clinical Lipidology

Original Article
Phenotypic variability in 4 homozygous familial hypercholesterolemia siblings compound heterozygous for LDLR mutations

https://doi.org/10.1016/j.jacl.2016.04.005Get rights and content

Highlights

  • Four FH siblings were found to be compound heterozygotes for 2 LDLR mutations.

  • They carried a known missense mutation and a minute deletion in exon 8/intron 8 junction.

  • The deletion causes an in-frame deletion of 17 amino acids in the LDLR protein.

  • Two siblings with a less severe phenotype were carriers of a rare missense apoB variant.

  • This rare missense apoB variant may have an LDL-lowering effect.

Background

Homozygous familial hypercholesterolemia is a rare clinical phenotype with a variable expression, which is characterized by extremely elevated plasma low-density lipoprotein (LDL), tendon and skin xanthomas, and a progressive atherosclerosis. In 95% of patients, homozygous familial hypercholesterolemia is due to mutations in low-density lipoprotein receptor (LDLR) gene, which abolish (receptor-negative) or greatly reduce (receptor-defective) LDLR function.

Objective

The objective of the study was the molecular and phenotypic characterization of 4 siblings with severe hypercholesterolemia.

Methods

The major LDL-related genes (LDLR, APOB, PCSK9, ANGPTL3, APOE, and APOC3) were sequenced. LDLR messenger RNA, isolated from leukocytes, was reverse transcribed and sequenced.

Results

The index cases were 24-year-old identical twin sisters with long-standing tendon xanthomas and high low-density lipoprotein cholesterol (LDL-C ∼10 mmol/L) but no coronary heart disease. They were carriers of 2 LDLR mutations: (1) a previously reported mutation [p.(G335S)] inherited from the mother who had LDL-C level within normal range; (2) a novel 24 bp deletion in exon 8/intron 8 junction inherited from the hypercholesterolemic (LDL-C 6.1 mmol/L) father. The deletion allele encodes an messenger RNA with a partial deletion of exon 8, whose translation product has an in-frame deletion of 17 amino acids [p.(Glu380_Gly396del)]. Family screening revealed that the 2 siblings of the twin sisters were also compound heterozygotes but had much lower LDL-C levels (8.2 and 7.1 mmol/L). The sequence of potential modifying genes showed that the 2 siblings and the mother of the twin sisters were heterozygous for a rare missense variant of apoB [p.(S2429T)], which might have an LDL-lowering effect.

Conclusions

We report a rare event of 4 siblings found to be compound heterozygotes for 2 LDLR gene mutations but showing a different phenotype severity. The less severely affected siblings were carriers of a rare apoB missense variant.

Introduction

Homozygous familial hypercholesterolemia (HoFH) is a rare clinical phenotype of severe low-density lipoprotein (LDL) hypercholesterolemia that is inherited in an autosomal dominant pattern with low-density lipoprotein receptor (LDLR) mutations. The high levels of low-density lipoprotein cholesterol (LDL-C) lead to cholesterol buildup in the skin and tendons right from early infancy. Cholesterol deposition in the intima of the arteries causes devastating atherosclerotic lesions and premature coronary heart disease (CHD). CHD and aortic stenosis (due to cholesterol deposition in aortic root and valve) may become detectable already during childhood. HoFH patients, if untreated, have a greatly reduced life expectancy, with a large proportion of patients suffering from fatal coronary insufficiency or myocardial infarction before the age of 20 years.1, 2, 3 More than 95% of patients with HoFH have mutations in both LDLR alleles.4, 5 Patients may carry 2 identical mutant alleles (true homozygotes) or 2 different mutant alleles (compound heterozygotes). The assay of LDLR activity in cultured skin fibroblasts is used to distinguish receptor-negative patients (<2% of residual LDLR activity) from receptor-defective patients (2%–30% LDLR activity). The residual LDLR activity is negatively correlated with the plasma level of LDL-C and the severity of clinical manifestations.4, 5, 6

Mutations in other genes, such as apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) or, rarely, the LDLR adaptor protein 1 (LDLRAP1; autosomal recessive hypercholesterolemia) are also found in patients with a clinical phenotype consistent with HoFH.4, 7, 8 Patients may also be double heterozygotes when they carry mutations in LDLR in combination with mutations in either APOB or PCSK9 gene.

From the clinical standpoint, the criteria commonly adopted for the diagnosis of dominant HoFH are based on LDL-C level ≥12 mmol/L, presence of cutaneous or tendon xanthomas in infancy (often associated with aortic stenosis), and hypercholesterolemia in both parents.2, 3, 9 The prevalence of dominant HoFH had been estimated to be 1:1.000.000 and the prevalence of heterozygous carriers 1:500.1 Recently, population studies in Denmark indicated that the prevalence of clinically defined heterozygous familial hypercholesterolemia (FH; based on Dutch Lipid Clinic criteria) was ∼1: 200.10 An extensive molecular study conducted in the Netherlands indicated that the prevalence of heterozygous carries of FH candidate gene mutations is ∼1:244.5 In view of these findings, the estimated prevalence of dominant HoFH would be between 1:160.000 and 1:360.000. The Dutch study also revealed that patients with molecularly defined HoFH have a variable clinical phenotype ranging from mild and/or moderate to the extremely severe.5 This variability may depend on the candidate gene involved and the type of mutation (eg, LDLR-negative vs LDLR-defective), as well as the contribution of variants in modifying genes and possibly lifestyle and dietary habits. In this context, one should consider the presence of rare variants in APOB gene (resulting in truncated apoBs or in single amino acid substitutions) that are the cause of familial hypobetalipoproteinemia type 1 (FHBL1; OMIM # 1077309).11 FHBL1 is an autosomal dominant disorder characterized by plasma levels of total cholesterol (TC), LDL-C, and ApoB > the 5th percentile, with a heterozygous frequency of 1:1000. Mutations in APOB gene associated with FHBL1 might contribute to the phenotypic variability in FH by exerting a strong LDL-lowering effect.12 This study was prompted by the observation of identical twin sisters in whom the clinical diagnosis of definite HoFH was made in the second decade of life when they were found to be compound heterozygotes for 2 mutations in the LDLR gene. The phenotypic expression of the disease in these twin sisters was more severe than that observed in their 2 siblings who were compound heterozygotes for the LDLR mutations but were also heterozygous for a rare missense variant of apoB with a possible LDL-lowering effect.

Section snippets

Plasma lipids

Plasma levels of TC, high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were determined by standard enzymatic techniques (Roche Diagnostics GmbH, Mannheim, Germany). LDL-C was calculated by Friedewald's formula. Lp(a) was measured by rate nephelometry (BN ProSpec; Siemens Healthcare Diagnostics, Italy).

Gene resequencing

Genomic DNA was extracted from peripheral blood leukocytes,6 and exon by exon sequencing of LDL-related genes (LDLR, PCSK9, APOB, APOE, ANGPTL3, and APOC3) and HDL-related

Clinical features of the probands

The probands were identical twin sisters (subjects III.2 and III.3 in Fig. 1) referred to the lipid clinic at the age of 24 years for the presence of xanthomas in the Achilles tendons, which had appeared during adolescence but had been overlooked until the age of 19 years when their presence was found to be associated with elevated levels of plasma cholesterol (reported TC ∼13 mmol/L). However, at that time, only dietary and lifestyle changes had been suggested, and no decision had been taken

Discussion

The index cases of the present study were 24-year-old identical twin sisters presenting a phenotype that was somewhat milder than most HoFH but nevertheless more severe than average for HeFH. Although their father displayed typical LDL-C concentration of HeFH, the LDL-C level in their mother did not suggest heterozygous FH. Furthermore, the phenotype in the 2 twin sisters' siblings was compatible with that of heterozygous FH. On first examination, we assumed that the twin sisters had a severe

Conclusions

The FH family described in this study reveals that a severe phenotype as HoFH may be overlooked for many years despite the presence of early clues for its clinical identification. The study also emphasizes that: (1) HoFH patients sharing the same LDLR mutations may present with a variable phenotypic expression in terms of plasma LDL-C level and clinical manifestations; (2) patients may not be fully compliant with the suggested treatment for several reasons such as putative “drug intolerance,” a

Acknowledgments

C.R., F.B., and M.P. performed the analysis of candidate genes and mutant mRNA characterization. P.T. supervised the molecular investigations. T.S. and F.S. were in charge of the patients management and clinical data collection. S.C. and S.B. contributed to the design of the study and wrote the article. All the authors read and approved the final version of the article.

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