Abstract
Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder, which leads to premature cardiovascular diseases. Microsomal triglyceride transport protein (MTP) inhibitors, such as lomitapide, offer a new therapeutic approach for treating these patients. We evaluated the lipid lowering (LL) efficacy of lomitapide according to several gene variants in MTP. Four clinically and/or molecularly defined HoFH patients were treated with lomitapide in addition to conventional high intensity LL therapy and regular lipoprotein apheresis. Two patients responded to the therapy, with a significant reduction of LDL cholesterol (LDL-C > 50%, hyper-responders). Sequencing of all exonic and intronic flanking regions of the MTP gene in all patients revealed 36 different variants. The hyper-responders to lomitapide shared six common variants: rs17533489, rs79194015, rs745075, rs41275715, rs1491246, and rs17533517, which were not seen in hypo-responders (reduction in LDL-C< 50%). We suggest that in HoFH variants in the MTP gene may impact on the therapeutic response to lomitapide, but this requires further investigation.
Keywords: Homozygous familial hypercholesterolemia, Microsomal transfer protein, Lomitapide, MTP gene
Introduction
Homozygous familial hypercholesterolemia (HoFH) is characterized by the following: extreme elevation of plasma low density lipoprotein cholesterol (LDL-C); accumulation of cholesterol in the corneas, eyelids, and extensor tendons; premature cardiovascular disease (CVD); and aortic valve disease1, 2). Recent evidence suggests that the condition may be as frequent as 1 in 160,000–300,000 individuals3). Severe and resistant cases are treated with LDL apheresis and high intensity statin based lowering (LL) drug therapy1). Furthermore, new therapies such as the use of proprotein convertase subtilisin kexin type-9 (PSCK9) inhibitors, mipomersen and lomitapide [microsomal triglyceride transfer protein (MTP) inhibitor] may be valuable in these patients4), but responsiveness in LDL-C can be variable and genetically determined.
Aim
In a prospective case series, we investigated whether the LL efficacy of lomitapide in HoFH patients was dependent on MTP gene variants.
Methods
Four patients (N1, N2, N3, and N4) diagnosed clinically and genetically (N1 and N2) as HoFH, were treated with diet and maximum doses of available LL drug combination (rosuvastatin plus ezetimibe plus colesevelam) and regular LDL apheresis sessions (every 5–10 days). Once the FDA approved lomitapide, 5 then 10 and 20–40 mg (as required) daily (one month apart) were added to usual, high intensity LL therapy and LDL apheresis sessions in all patients. To avoid adverse gastrointestinal events and vitamins deficiency, the restricted low-fat diet (< 20% of energy from fat) was introduced and vitamin E 268 mg (400 IU, Solgar®) and omega 3-6-9 (Solgar®) were added. Patients with reductions in LDL-C of > 50% and approaching LDL-C therapeutic targets at follow-up were classified as hyper-responders, and those with reductions in LDL-C of < 50% and still requiring LDL apheresis sessions to improve LDL-C were classified as hyporesponders. The diagnosis of HoFH in our patients was based on the Cuchel et al criteria5); see below. Sequencing of the LDLR gene in N1 and N2 patients revealed mutations leading to altered protein sequence and/or function [patient N1: combined heterozygosity c.1448G > A, (p.Trp483X) and c.1646G > A (p. Gly549Asp), which resulted in altered LDLR function according to the prediction tool polyphen (score: 0.999; probably damaging); patient N2 was found homozygous for c.1285G > A (p.Val429Met) resulting in < 2% of LDLR function]. In patients N3 and N4, the DNA testing for FH was not carried out. The duration of lomitapide treatment was 14, 13, 7 and 6 months in patients N1, N2, N3, and N4, respectively. Demographic and clinical data of all patients are presented in Table 1. MTP gene analysis was performed in all patients. Briefly, genomic DNA was isolated from whole blood using the High Pure PCR Template Preparation Kit (Roche). Investigation of genetic changes in the 26 exons and flanking intronic regions of the MTP gene was performed using PCR and direct sequencing of PCR products. The PCR product was sequenced using the ABI3500 genetic analyzer. Sequence analysis was performed by the Seqscape module using ENSG00000144285/ENST00000303395 as reference DNA sequence.
Table 1. Demographic and clinical characteristics and plasma lipid and lipoprotein concentrations before treatment in all the four patients reported.
| Patient, Sex | Age | BMI | Years of LDL apheresis | CVD | Lipid profile (in mg/dl) |
|---|---|---|---|---|---|
| N1, Male combined heterozygosity c.1448G > A, c.1646G > A |
25 | 25 | 19 | Mild carotid stenosis | TC: 1000 TG: 150 HDL: 32 LDL: 900 Lp(a): 19 |
| N2, Male homozygous c.1285G > A |
22 | 25 | 10 | AVR, Carotid stenosis | TC: 1100 TG: 130 HDL: 33 LDL: 950 Lp(a): 5.5 |
| N3, Female Mutation status not available |
47 | 28 | 2 | CABG + AVR, Carotid surgery | TC: 1000 TG: 110 HDL: 18–30 LDL: 900 Lp (a): 16 |
| N4, Male Mutation status not available |
56 | 27 | 12 | CABG x2, PCIx2, AVR, Carotid Surgery | TC: 800 TG: 110 HDL: 50 LDL: 780 Lp (a): 36 |
To convert cholesterol values (TC, HDL, and LDL) from mg/dl to mmol/l, multiply by 0.0259, and, for TGs, multiply by 0.0113. TC indicates total cholesterol, TG indicates triglycerides, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein. Lp (a) indicates lipoprotein (a), CABG indicates coronary artery bypass grafting; AVR indicates aortic valve replacement; PCI indicates percutaneous coronary intervention.
Because no information is available for most of the MTP gene variants in the literature, the hypothetical approach was to define common gene variants that were associated with response to lomitapide.
Clinical Criteria for the Diagnosis of HoFH
The diagnosis of HoFH in patients N3 and N4 were made on the basis of clinical criteria suggested by the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society 5) (untreated LDL-C of > 13 mmol/L (500 mg/dl), cutaneous or tendon xanthomata before 10 years of age, and both parents with phenotypic heterozygous (He) FH, which we diagnosed according to the Dutch Lipid Clinic Network criteria.6)).
Results
Patients with the clinical criteria of HoFH (N3 and N4) had total cholesterol 1,000 and 800 mg/dl, respectively before any treatment of (Table 1), tendon xanthomata before 10 years of age, and both parents with definite HeFH diagnosed by Dutch Lipid Clinic Network criteria).
Both hyper-responders were treated with lomitapide 10 mg daily and the hypo-responders with 40 mg/daily (N1) and 30 mg/daily (N3). Patients N2 and N4 showed the highest reduction in plasma LDL-C concentration after the addition of lomitapide and were classified as hyper-responders (Table 2). Patients N1 and Patient N3 responded to lomitapide treatment with a lower reduction in LDL-C (Table 2) and required additionally LDL apheresis sessions to reach the treatment target and were classified as hyporesponders7). As shown in Table 3, 36 different MTP gene variants were found in all patients. The hyperresponders (N2 and N4) shared six MTP gene variants (rs17533489, rs79194015, rs745075, rs41275715, rs1491246, and rs17533517). There was no sharing of these variants in hypo-responders (N1 and N3). Five variants (rs991811, rs2306985, rs881981, rs2718684, and rs3816873) were common to 3 or to 4 patients (hyper-/hypo-responders), and thus, were not associated with LL therapeutic response to lomitapide. Patients N1 (hypo-responder) and N2 (hyperresponder) had seven common variants (rs17029213, rs17029215, rs34734558, rs41275719, rs371030218, rs34883891, and rs7667001), and thus, were not associated with the LL response. The variants exclusive to one hypo- or hyper-responder (Patient N1: rs2306984, rs2298747, rs3792681, and rs170291; Patient N2: rs17029189, rs113557405, rs112568939, rs113337987, and rs112407688; Patient N3: rs3833621, rs2306986, rs3792683, rs982424, and rs2255119; and Patient N4: rs61733139, rs114681504, rs1491244, and rs74542928) suggest the LL effect of lomitapide only in a particular patient, Table 4.
Table 2. Interval mean values for plasma lipid and lipoprotein concentrations before and mean values (patients N2 and N4) and interval mean values (patients N1 and N3) after lomitapide treatment in the HoFH patients.
| Patient | Before | After | mg/dl Changes |
% Changes |
|---|---|---|---|---|
| N1 | ||||
| TC | 527 ± 72 | 329 ± 49 | −198 | −38 |
| TG | 128 ± 51 | 64 ± 16 | −64 | −50 |
| HDL | 30 ± 6 | 32 ± 5 | 2 | 7 |
| LDL | 472 ± 65 | 305 ± 51 | −167 | −35 |
| N2 | ||||
| TC | 388 ± 23 | 177 ± 15 | −211 | −55 |
| TG | 142 ± 35 | 102 ± 55 | −40 | −28 |
| HDL | 32 ± 2 | 33 ± 2 | 1 | 3 |
| LDL | 330 ± 25 | 114 ± 15 | −216 | −65 |
| N3 | ||||
| TC | 295 ± 24 | 236 ± 47 | −59 | −20 |
| TG | 105 ± 38 | 69 ± 19 | −36 | −34 |
| HDL | 27 ± 3 | 25 ± 4 | −2 | −7 |
| LDL | 220 ± 27 | 196 ± 46 | −24 | −11 |
| N4 | ||||
| TC | 322 ± 40 | 180 ± 25 | 142 | −45 |
| TG | 114 ± 13 | 70 ± 22 | −34 | −39 |
| HDL | 40 ± 2 | 45 ± 6 | 5 | 12 |
| LDL | 259 ± 38 | 118 ± 30 | −141 | −54 |
Before indicates the TC, TG, HDL and LDL values before adding lomitapide to regularly treatment with lipid lowering drugs and LDL apheresis sessions. In patients N2 and N4 “After” indicates mean values for blood lipids with lomitapide treatment and no LDL apheresis sessions and in patients N1 and N3 “After” indicates interval mean values after adding lomitapide and with LDL apheresis sessions. All data were expressed as mg/dl, mean ± SD. The mean data were based on last 5 measurements before and Lomitapide administration. To convert cholesterol values (TC, HDL, and LDL) from mg/dl to mmol/l, multiply by 0.0259, and, for TGs, multiply by 0.0113. TC indicates total cholesterol, TG indicates triglycerides, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein.
Table 3. MTP Gene Variants of all four Patients.
| Patient N1 | Patient N2 | Patient N3 | Patient N4 |
|---|---|---|---|
| rs7667001 | rs7667001 | rs3833621 | rs61733139 |
| rs3792681 | rs3816873 | rs2306986 | rs3816873 |
| rs991811 | rs991811 | rs3816873 | rs991811 |
| rs170291 | rs17029189 | rs991811 | rs2306985 |
| rs34883891 | rs113557405 | rs3792683 | rs17533489 |
| rs2306984 | rs34883891 | rs982424 | rs79194015 |
| rs2306985 | rs2306985 | rs2306985 | rs17533517 |
| rs17029213 | rs17029213 | rs2718684 | rs114681504 |
| rs17029215 | rs17533489 | rs881981 | rs745075 |
| rs34734558 | rs79194015 | rs2255119 | rs41275715 |
| rs2298747 | rs17533517 | rs1491246 | |
| rs2718684 | rs17029215 | rs1491244 | |
| rs41275719 | rs34734558 | rs881981 | |
| rs881981 | rs112568939 | rs74542928 | |
| rs371030218 | rs113337987 | ||
| rs745075 | |||
| rs41275715 | |||
| rs2718684 | |||
| rs1491246 | |||
| rs41275719 | |||
| rs881981 | |||
| rs371030218 | |||
| rs112407688 |
Table 4. MTP gene variants categorized by patient or groups of patients.
| Patients: N1, N2, N3, N4 |
| rs991811, rs2306985, rs881981 |
| Patients: N1, N2, N3, |
| rs2718684 |
| Patients: N2, N3, N4 |
| rs3816873 |
| Patients: N1, N2 |
| rs17029213, rs17029215, rs34734558, rs41275719, rs371030218, rs34883891, rs7667001 |
| Patients: N2, N4 |
| rs17533489, rs79194015, rs745075, rs41275715, rs1491246, rs17533517 |
| Patient N1 |
| rs2306984, rs2298747, rs3792681, rs170291 |
| Patient N2 |
| rs17029189, rs113557405, rs112568939, rs113337987, rs112407688 |
| Patient N3 |
| rs3833621, rs2306986, rs3792683, rs982424, rs2255119 |
| Patient N4 |
| rs61733139, rs114681504, rs1491244, rs74542928 |
Discussion
MTP inhibitors offer a new therapeutic approach that has been FDA and EMA approved for HoFH patients; however, the reduction in LDL-C is variable7). Careful attention to diet and safety monitoring (liver function and gastrointestinal complains) is necessary when using this agent7). Raper et al.8) reported a HoFH patient treated for 5 years with lomitapide who achieved a marked reduction in LDL-C concentration, reaching the desirable target level of 70 mg/dl. Cuchel et al.9) reported overall reductions in LDL-C from baseline by 50% with the median dose of lomitapide of 40 mg/daily. Furthermore, of the 23 patients who completed the trial 16 achieved the therapeutic goals, with LDL-C of < 100 mg/dl achieved by 8. In our study, we treated four HoFH (two with documented < 2% of LDLR function) patients with lomitapide for a prolonged period of 14 months. Two patients had an extremely good response to lomitapide, with LDL-C reductions of 54% and 65% (hyper-responders), compared with two others with LDL-C reductions of < 35% (hypo-responders). The reasons for the excellent response of the former two are unknown. All patients were given the similar diet, had desirable body mass index, and were treated with the same LL drugs and dose. That hyper-responders to lomitapide required lower doses of this drug than hypo-responders provides further internal consistency to our case definition and study hypothesis. One explanation could be that certain LDLR mutations are associated with better LL response to lomitapide. Two of our patients had documented non-functioning LDLR, yet one of them was a hyper-responder, suggesting a mechanism involving decreased hepatic secretion of VLDL apoB and the subdequent production of LDL apoB10). Ledmyr et al.11) reported that the rs3816873 variant influences the interaction between MTP and apoB and that rs3816873 128Thr allele is associated with reduced MTP expression and function. We found the rs3816873 variant in three patients, all with CVD and/or aortic valvular disease with a variable response to LL effect of lomitapide. Hagan et al.12) in experimental study reported that C-allele of the MTP-164T > C variant is homologous to a putative sterol response element (SRE) binding site and is up-regulated by cholesterol levels. In states of high cholesterol levels, the modified SRE likely binds a new sterol regulatory element binding protein (SREBP) and up-regulates MTP expression. These observations suggest that MTP gene expression is differently regulated by high and low cholesterol levels. In our patients, the cholesterol level before lomitapide treatment was very high. However, other than described by Hogan et al.10), MTP variant could also be an antagonist between cholesterol levels and MTP.
Over the last decade, significant efforts have been made to improve personalized medicine, with enhanced effectiveness and safety of new drugs. This can be facilitated by testing particular gene variants involved in drug metabolism, such as (CYP450), or regarding hypolipidemic drugs variants (e.g., CETP, apoE, LDL receptor) that regulate lipid metabolism. With lomitapide, the encoding MTP gene variant may improve the management of such difficult patients. Reliable inferences based on data from only four patients are questionable. However, the differences reported between hypo- and hyper-responders in our investigation were sufficiently clear to allow us to generate a good hypothesis that can be tested in future studies.
We suggest that the six SNPs rs17533489, rs79194015, rs745075, rs41275715, rs1491246, and rs17533517 (C/T allele) of the MTP gene recorded after sequencing of the whole MTP gene may influence the LL response to lomitapide because they were only found in hyper-responders. The MTP gene variants can potentially influence the LL effect of lomitapide or may be involved in a more complex network of gene–gene or gene–environment interaction, in which certain variants may influence the function of the MTP protein. Moreover, individually, the effect of the gene variants studied on the LL response to lomitapide is probably small, but collectively, it could have a significant stacking effect on the therapeutic response to this drug. Further studies are required to elucidate the role of these gene variants.
The small number of the patients studied is one of the limitations of our work. However, this is owing to an extremely rare prevalence of HoFH and the involvement of only one center. According to historical prevalence data (one in a million), there are 900 individuals in the European region; however, according to extrapolation from recent Danish general population evaluated by Nordestgaard et al.13) (∼1 in 160,000), there are 5,630 individuals in the European region. Furthermore, the diagnosis of HoFH is still underdiagnosed.
Conclusion
The absence of convincing explanation regarding the different response of patients under the same therapeutic protocol strengthens our hypothesis of a potential contribution of MTP variants in the LL effect of lomitapide. This case series is only a hypothesis, but the findings suggest that further investigation is warranted to establish whether MTP gene variants determine the therapeutic response of LDL-C to lomitapide in FH and that knowing the likely response based on gene score could enrich the decision making and physician and patient expectations in the future.
Conflicts of Interest
None.
References
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