Genetic Analysis in a Taiwanese Cohort of 750 Index Patients with Clinically Diagnosed Familial Hypercholesterolemia

Abstract

Aim: Familial hypercholesterolemia (FH) is underdiagnosed in most countries. The genetic heterogeneity of FH requires an algorithm to efficiently integrate genetic testing into clinical practice. We aimed to report the spectrum of genetic mutations from patients with clinically diagnosed FH in Taiwan.

Methods: Patients with LDL-C＞190 mg/dL or those with probable or definite FH according to the Taiwan Lipid Guidelines underwent genetic testing. Samples from 750 index patients from the Taiwan FH registry were screened using custom-made mass spectrometry, followed by targeted next generation sequencing (NGS) and/or multiplex ligation-dependent probe amplification (MLPA) if found negative.

Results: The mean age of the patients was 52.4±15.1 years and 40.9% were male. Mutations were detected in 445 patients (59.3%). The distribution of mutations was as follows: LDLR (n=395), APOB (n=58), PCSK9 (n=0), and ABCG5 (n=3). The most common mutations were APOB c.10579 C＞T (p.R3527W) (12.6%), LDLR c.986 G＞A (p.C329Y) (11.5%), and LDLR c.1747 C＞T (p.H583Y) (10.8%). LDLR c.1187-10 G＞A (IVS 8-10) and APOB c.10580 G＞A (p.R3527Q) were detected using targeted NGS in Taiwan for the first time. Four novel mutations were identified, including LDLR c.1060＋2 T＞C (IVS 7＋2), LDLR c.1139 A＞C (p.E380A), LDLR c.1322 T＞C (p.A431T)＋c.1867 A＞G (p.I623V), and ABCG5 c.1337 G＞A (p.R447Q).

Conclusion: LDLR and APOB, but not PCSK9, mutations were the major genetic causes of FH. Four novel mutations in LDLR or ABCG5 were identified. This genetic screening method using mass spectrometry, targeted NGS, and MLPA analysis provided an efficient algorithm for genetic testing for clinically diagnosed FH in Taiwan.

Keywords: Cholesterol; Familial hypercholesterolemia; Gene mutation; Lipids; Mass spectrometry; lipoprotein metabolism.

Fig.1. The algorithm for the genetic diagnosis of patients with definite or probable familial hypercholesterolemia (FH) enrolled in this study

All patients were initially screened by a custom-made mass spectrometry, which can simultaneously detect 68 known FH mutations in Taiwan. If its result was negative and the LDL-C level was ＞250 mg/dL, a more comprehensive targeted next generation sequencing (NGS), was performed to detect any novel variations not included in the mutation panels of mass spectrometry. If the targeted NGS was still negative, multiplex ligation-dependent probe amplification (MLPA) analysis was necessary to detect LDLR large gene rearrangements.

Fig.2. Genetic diagnosis of a family with LDLR mutation

The index patient was a 4-year-old boy that presented with xanthoma over both ankles since birth. Genetic testing showed three LDLR mutations detected by mass spectrometry. Based on the Sanger sequencing analysis of DNA collected from his family, the LDLR c.1322 T＞C (p.A431T)＋LDLR c.1867 A＞G (p.I623V) are in one allele and LDLR c.1747 C＞T (p.H583Y) is in the other allele. CVD, cardiovascular disease; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride.

Fig.3. Family pedigree of LDLR c.1060＋2 T＞C (T＞C at IVS 7＋2)

The index patient was genetically diagnosed as LDLR c.1060+2 T＞C (T＞C at IVS 7+2), which was a novel intronic mutation of LDLR. The LDLR c.1060＋2 T＞C mutations co-segregated well with LDL-C levels in this family study. CVD, cardiovascular disease; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride.

Fig.4. Family pedigree of LDLR c.1139 A＞C (p.E380A)

The index patient was genetically diagnosed as LDLR c.1139 A＞C (p.E380A), which was novel and has not been reported before. However, this genotype did not co-segregate well with the phenotype of hypercholesterolemia in this family. CVD, cardiovascular disease; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride.