Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia).

BACKGROUND AND AIMS: Familial dysbetalipoproteinemia (FDBL) is a rare inborn lipid disorder characterized by the formation of abnormal triglyceride- and cholesterol-rich lipoproteins (remnant particles). Patients with FDBL have a high risk for atherosclerotic disease. The effect of PCSK9 inhibition on lipoproteins and its subfractions has not been evaluated in FDBL.
METHODS: Three patients (65±7 years, 23±3 kg/m2, 2 females) with FDBL (diagnosed by isoelectrofocusing) and atherosclerosis (coronary and/or cerebro-vascular and/or peripheral arterial disease) resistant or intolerant to statin and fibrate therapy received evolocumab (140mg every 14 days). In addition to a fasting lipid profile (preparative ultracentrifugation), apoB and cholesterol concentrations were determined in 15 lipoprotein-subfractions (density gradient ultracentrifugation; d 1.006-1.21g/ml) before and after 12 weeks of evolocumab treatment. Patients with LDL-hypercholesterolemia (n = 8, 56±8 years, 31±7 kg/m2) and mixed hyperlipidemia (n = 5, 68±12 years, 30±1 kg/m2) also receiving evolocumab for 12 weeks were used for comparison.
RESULTS: All patients tolerated PCSK9 inhibition well. PCSK9 inhibitors reduced cholesterol (29-37%), non-HDL-cholesterol (36-50%) and apoB (40-52%) in all patient groups including FDBL. In FDBL, PCSK9 inhibition reduced VLDL-cholesterol and the concentration of apoB containing lipoproteins throughout the whole density spectrum (VLDL, IDL, remnants, LDL). Lipoprotein(a) was decreased in all patient groups to a similar extent.
CONCLUSIONS: This indicates that the dominant fraction of apoB-containing lipoproteins is reduced with PCSK9 inhibition, i.e. LDL in hypercholesterolemia and mixed hyperlipidemia, and cholesterol-rich VLDL, remnants and LDL in FDBL. PCSK9 inhibition may be a treatment option in patients with FDBL resistant or intolerant to statin and/or fibrate therapy.

Introduction

Familial dysbetalipoproteinemia (FDBL) is an uncommon form of mixed hyperlipidemia characterized by the accumulation of triglyceride- and cholesterol-rich lipoproteins (remnant particles) in plasma [1]. FDBL is caused by mutations in apoE, typically homozygosity for apoE2, which represents a mutant form of apoE, an apoprotein relevant for the metabolism of triglyceride- and cholesterol-containing lipoproteins [2]. However, homozygosity for apoE2 is not sufficient to induce the characteristic dyslipidemia, dysbetalipoproteinemia (formerly known as type III hyperlipidemia). The development of dysbetalipoproteinemia follows a second hit (environmental factors, other genetic defects, comorbidities). If this secondary factor can be eliminated, lipid profiles may become “normal” again. If dysbetalipoproteinemia is present, it is associated with premature cardiovascular disease [3, 4]. The pathophysiology behind this dyslipidemia relates to the fact that apoB and apoE can bind to the LDL-receptor. Remnants are cleared from the plasma due to the interaction of apoE with the LDL-receptor. ApoE2 has the lowest affinity to the LDL-receptor of all apoE isoforms. This is why in E2/E2 patients remnant particles accumulate in plasma [5]. Although mixed hyperlipidemia (elevated triglycerides and elevated cholesterol) is the typical dyslipidemia in these patients, some may also present with other forms of dyslipidemia such as isolated hypertriglyceridemia or hypercholesterolemia [1].

The clinical management of FDBL is complicated by the fact that the determination of LDL-cholesterol is not reliable as remnant particles may be classified as VLDL or LDL depending on the triglyceride load and method of determination. Therefore, non-HDL-cholesterol is used to guide therapy [1]. Treatment of dysbetalipoproteinemia can be challenging as standard lipid lowering therapies such as statins and fibrates are sometimes not very effective or cannot be tolerated. Niacin, which has been used in the past, is not available anymore in many countries [6]. PCSK9-inhibitors, a new potent treatment option for lowering LDL-cholesterol [7, 8], have not been evaluated in patients with FDBL. PCSK9 is an important regulator of LDL receptor expression but has a number of additional functions as recently described [9]. PCSK9 inhibitors have a strong effect on the lipid profile primarily reducing LDL-cholesterol (-50 to -60%) but also reducing lipoprotein(a) (-20 to -30%). They have little effect on triglyceride levels (-12 to -17%) [10, 11]. Similar to statins and ezetimibe, it was shown that PCSK9-inhibition results in ASCVD risk reduction, and risk reduction is closely related to LDL-cholesterol reduction [7, 8].

For clinical purposes, apoB-containing lipoproteins are usually divided into VLDL and LDL, although all lipoprotein fractions including the LDL fraction represent continua of particles with variable amounts of triglycerides and cholesterol. When gradient ultracentrifugation is used to subdivide the non-VLDL fraction, 15 subfractions are isolated with IDL, LDL and HDL representing subfractions 1–4, 5–11, and 12–15 respectively [12, 13]. Small dense LDL (subfractions 9–11) and probably also large buoyant LDL (subfractions 5–6) are more atherogenic than intermediate dense LDL (subfractions 7–8). Thus, the determination of LDL subfractions may provide additional information about the atherogenic potential of a lipid profile. Evaluating lipoprotein subfractions is particularly interesting in conditions where the concentration of a broad spectrum of particles is increased, as it is the case in FDBL.

We therefore evaluated the effect of PCSK9-inhibition on lipoprotein subfractions in patients with FDBL and compared it to the effect in patients with LDL-hypercholesterolemia and patients with mixed hyperlipidemia (not FDBL).

Patients and methods

In this single center study patients with FDBL (apoE2 homozygosity; n = 3), mixed hyperlipidemia (apoE3 or apoE4; n = 5), and isolated LDL-hypercholesterolemia (familial hypercholesterolemia or other forms of isolated hypercholesterolemia; apoE3 or apoE4; n = 8) were recruited from our registry on patients receiving PCSK9-inhibitors. We included all patients with apoE2/E2 phenotype and selected consecutive patients with isolated hypercholesterolemia and mixed hyperlipidemia. All patients had the indication for treatment with PCSK9-inhibitors according to German regulatory standards (not reaching treatment goals recommended by the European guidelines despite maximally tolerated oral lipid-lowering therapy) [14] and received evolocumab 140 mg sc every 2 weeks.

Patients with FDBL were characterized by the typical lipid profile with elevated total cholesterol and elevated triglycerides. In addition, VLDL-cholesterol to VLDL-triglyceride ratio was elevated (>1). All patients suffered from ASCVD, thus either cerebrovascular disease (CVD), coronary artery disease (CAD) or peripheral arterial disease (PAD) and were statin-intolerant with one patient also not tolerating ezetimibe.

Patients with mixed hyperlipidemia all suffered from metabolic syndrome but none from diabetes mellitus. Compared to patients with FDBL, these patients had slightly lower total cholesterol and slightly lower triglyceride concentrations, resulting in an almost identical cholesterol to triglyceride ratio. However in contrast to the patients with FDBL, most of the cholesterol was associated with the LDL fraction and not with the VLDL fraction, resulting in a very different VLDL-cholesterol to VLDL-triglyceride ratio. All patients suffered from ASCVD. Three of the patients did not tolerate statin or ezetimibe, while two were treated with high dose statin and ezetimibe.

Six of the patients with hypercholesterolemia suffered from heterozygous familial hypercholesterolemia (Dutch Lipid Clinic Network (DLCN) Score: probable familial hypercholesterolemia), while 2 did not (DLCN-Score: unlikely or possible familial hypercholesterolemia). All patients had severely elevated total cholesterol because of elevated LDL-cholesterol. All but one patient (patient with heterozygous familial hypercholesterolemia and strong positive family history for atherosclerotic disease) suffered from ASCVD. Four of the patients did not tolerate statins and one did also not tolerate ezetimibe. One patient was treated with additional colesevelam.

The study protocol was approved by the ethics committee of the University of Munich (Protocol number 17–780) and all patients signed informed consent. The study abide by the Declaration of Helsinki principles.

Cholesterol, triglycerides, lipoprotein(a) and apoB concentrations were determined in fasting plasma and different lipoprotein fractions enzymatically before initiation of PCSK9-inhibitor therapy and after 3 months of treatment using an automated clinical chemistry analyser (Response 910, DiaSys, Flacht, Germany). ApoE phenotype was determined once in each subject using isoelectrofocusing as described before [15].

Lipoprotein subfractions were isolated by preparative ultracentrifugation (18 hours, d = 1.006 g/mL, 50000 rpm, 4°C, Beckmann Ti 50.4 rotor). In the supernatant, VLDL-cholesterol and VLDL-triglycerides were measured. In the infranatant subfractions were isolated by isopycnic density gradient ultracentrifugation, as described previously [12, 13]. Densities were measured by a precision density meter (Anton Paar DMA 38, Graz, Austria). Ultracentrifugation was performed in a Beckmann SW 40 Ti rotor (Palo Alto, CA) at 40 000 rpm for 48 hours at 15°C. A total of 15 subfractions (SF) were isolated with the following density intervals: SF 1–4, 1.006–1.019; SF 5, 1.020–1.024; SF 6, 1.025–1.029; SF 7, 1.030–1.034; SF 8, 1.035–1.040; SF 9, 1.041–1.047; SF 10, 1.048–1.057; SF 11, 1.058–1.066; SF 12–15, 1.067–1.210 g/mL), with IDL, LDL and HDL representing SF 1–4, 5–11, and 12–15 respectively. Lipoprotein(a) is usually found in SF 11–13; in subjects with elevated lipoprotein(a), the fraction containing the smallest LDL subfraction may therefore contain substantial amounts of cholesterol transported on lipoprotein(a). In all subfractions cholesterol and apoB were determined.

Statistical evaluation: No formal statistical evaluation was performed as group sizes are too small to execute meaningful statistical testing. Descriptive data are given as means ± SD.

Results

The characteristics of the study participants are shown in Table 1. Most patients received evolocumab because they were either partially or completely statin intolerant. ASCVD was present in all except one subject. Metabolic syndrome was much more common in patients with mixed dyslipidemia. Background lipid lowering therapy was not changed between baseline evaluation and the analysis after 3 months. All subjects tolerated PCSK9 inhibition well and finished the study.

Table 1

Characteristics of study participants.

	Familial dysbetalipoproteinemia	Mixed hyperlipidemia	Hyper-cholesterolemia
n	3	5	8
Male/female (n)	1/2	3/2	2/6
Age (years) ± SD	65 ± 7	68 ± 12	56 ± 8
BMI (kg/m2) ± SD	23 ± 3	30 ± 1	31 ± 7
Metabolic syndrome	1	5	4
CAD (%)	66.7	100	62.5
CVD (%)	33.3	80	25
PAD (%)	0	20	0
High dose statin therapy (n)	0	2	3
Any statin therapy (n)	0	2	4
Ezetimibe therapy (n)	2	2	7
Other lipid therapy (n)	0	0	1

BMI, body mass index; CAD, coronary artery disease; CVD: cerebral-vascular disease; PAD: peripheral arterial disease.

The effects on lipids, apoB and lipoprotein(a) are shown in Table 2, revealing considerable differences between the different patient groups. While the effect on total cholesterol, non-HDL-cholesterol, and apoB is comparable, remarkable differences are seen for triglycerides, LDL-cholesterol and VLDL-cholesterol as well as VLDL-triglycerides indicating a reduction of all apoB containing lipoproteins in FDBL and a predominant reduction of LDL in patients with hypercholesterolemia and mixed hyperlipidemia. In FDBL patients VLDL-cholesterol is reduced much more than VLDL-triglycerides indicating that smaller cholesterol-rich particles are preferentially removed, while the concentration of more typical VLDL is much less affected. In contrast, in patients with hypercholesterolemia and mixed hyperlipidemia, VLDL-cholesterol and VLDL-triglycerides are reduced to a similar extent (Fig 1).

Table 2

Lipid parameters in different patient populations before and during (12 weeks) of therapy with PCSK9 inhibitors.

	Familial dysbetalipoproteinemia			Mixed hyperlipidemia			Hypercholesterolemia
	before	during	(%)a	before	during	(%)a	before	during	(%)a
total cholesterol (mmol/l)	8.64 ± 4.76	5.46 ± 2.38	- 37	7.34 ± 1.63	4.34 ± 1.45	- 40	6.93 ± 2.38	4.94 ± 1.68	- 29
LDL cholesterol (mmol/l)	2.02 ± 0.44	1.30 ± 0.31	- 36	3.88 ± 1.27	1.60 ± 1.14	- 59	4.91 ± 2.07	2.95 ± 1.55	- 40
HDL cholesterol (mmol/l)	1.24 ± 0.21	1.14 ± 0.28	- 8	1.11 ± 0.18	1.22 ± 0.23	+ 10	1.66 ± 0.80	1.58 ± 0.67	- 4
non-HDL cholesterol (mmol/l)	7.40 ± 4.94	4.29 ± 2.61	- 42	6.23 ± 1.60	3.13 ± 1.40	- 50	5.28 ± 2.30	3.36 ± 1.63	- 36
total triglycerides (mmol/l)	4.01 ± 2.14	3.50 ± 2.61	- 36	3.39± 0.86	3.27 ± 2.78	- 3	1.11 ± 0.31	1.38 ± 0.46	+ 25
VLDL cholesterol (mmol/l)	5.38 ± 4.53	3.0 ± 2.84	- 44	2.35 ± 0.78	1.53 ± 1.50	- 35	0.34 ± 0.31	0.41 ± 0.28	+ 19
VLDL triglycerides (mmol/l)	3.85 ± 2.14	3.29 ± 2.58	- 14	3.21 ± 0.73	3.14 ± 2.72	- 2	0.82 ± 0.38	1.19 ± 0.56	+ 45
Apolipoprotein B (mg/dl)	91 ± 32	52 ± 11	- 42	144 ± 34	70 ± 25	- 52	141 ± 49	85 ± 33	- 40
Lipoprotein(a) (mg/dl)	44 ± 31	35 ± 29	- 20	44 ± 79	26 ± 47	- 41	67 ± 54	48 ± 40	- 28

Refers to % change observed during therapy with PCSK9 inhibitors.

Fig 1

VLDL-cholesterol (panel A) and VLDL-triglycerides (panel B) before (baseline, filled columns) and after 12 weeks (striped columns) of PCSK9 inhibition.

Different colors indicate different patient groups. Shown are means ± SD; green: FDBL, familial dysbetalipoproteinemia; red: mixed HLP, mixed hyperlipidemia; blue: HCH, hypercholesterolemia.

The effect on lipoprotein subfractions (IDL-HDL) is shown in Fig 2 (cholesterol) and Fig 3 (apoB). In hypercholesterolemia and mixed hyperlipidemia, the fractions containing most of apoB and cholesterol are fractions 5–11, representing LDL, while in FDBL, cholesterol and apoB are more equally distributed between fractions 1–11, which contain IDL and LDL. PCSK9 inhibition reduces fractions 5–11 in hypercholesterolemia and 1–11 in mixed hyperlipidemia and FDBL.

Fig 2

Cholesterol content (means) in different lipoprotein subfractions before (baseline, solid line) and after 12 weeks (dashed line) of PCSK9 inhibition.

Different colors indicate different patient groups. HCH, hypercholesterolemia; mixed HLP, mixed hyperlipidemia; FDBL, familial dysbetalipoproteinemia.

Fig 3

ApoB content (means) in different lipoprotein subfractions before (baseline, solid line) and after 12 weeks (dashed line) of PCSK9 inhibition.

Different colors indicate different patient groups. HCH, hypercholesterolemia; mixed HLP, mixed hyperlipidemia; FDBL, familial dysbetalipoproteinemia.

Discussion

PCSK9 inhibition reduces the plasma concentration of all apoB-containing lipoproteins except large, triglyceride-rich VLDL. This is true in patients with isolated LDL-hypercholesterolemia, with mixed hyperlipidemia and with familial dysbetalipoproteinemia. This observation is consistent with a dramatically increased LDL-receptor activity resulting in the uptake of all apoB-containing lipoproteins, except when the apoB receptor binding region is not accessible, as is the case for large triglyceride-rich VLDL [16].

It is noteworthy that in both hypertriglyceridemic patient groups (familial dysbetalipoproteinemia and mixed hyperlipidemia) VLDL-cholesterol is decreased to a larger extent than VLDL-triglycerides, indicating that there might be distinct groups of particles, some of which can be reduced by an increased LDL-receptor activity while others cannot be reduced. In absolute terms, VLDL-cholesterol is much higher at baseline in FDBL than in mixed hyperlipidemia. This is consistent with the known lipoprotein abnormalities in FDBL, where mostly the concentration of remnant particles is increased. In mixed hyperlipidemia (not FDBL) presumably fewer lipoproteins floating in the VLDL-fraction are removed by an increased LDL-receptor activity, because most of the triglyceride-rich lipoproteins observed in mixed hyperlipidemia are “typical” VLDL with little affinity to the LDL-receptor.

It can be assumed that the “anti-atherosclerotic” potential of PCSK9-inhibition is similar in FDBL, mixed hyperlipidemia and hypercholesterolemia as the concentration of apoB-containing lipoproteins is reduced to a similar extent. While in patients with mixed hyperlipidemia and hypercholesterolemia the reduction of apoB-containing lipoproteins occurs primarily in the LDL-fraction, a much broader range of particles, though mostly VLDL, is reduced in FDBL. This is confirmed by the effect of PCSK9-inhibition on lipoprotein subfractions (Figs 2 and 3). Cholesterol and apoB are reduced over the whole range of apoB-containing lipoproteins in FDBL. Although all fractions are reduced in proportion, in absolute terms, the most prominent fraction is reduced the most. The reduction of apoB containing lipoproteins also extends to lipoprotein(a), which was reduced in all 3 patient groups. It can be assumed that this contributes to the risk reduction, although the relative importance is still controversial [17].

The effect of statins, fibrates, ezetimibe and PCSK9-inhibititors on lipoprotein subfractions has been studied in the past, with no data (to our knowledge) in patients with FDBL [18-31]. Statins predominantly decrease intermediate dense LDL with some discordant results (some showing similar reductions in larger and smaller LDL while others show a predominant effect on intermediate dense LDL). Fibrates generally induce a shift in subfraction distribution (from small dense LDL towards less dense LDL) [24, 29]. A reduction of all LDL-subfraction was also seen during PCSK9-inhibititor treatment [30, 31].

Theoretically, the data are also consistent with a model in which PCSK9-inhibition primarily induces an elevated uptake of intermediate dense LDL particles and at the same time enhances the delipidation of larger lipoproteins to LDL particles. However, from a physiological point of view it is more likely that the increased activity of the LDL-receptor results in a direct uptake of a wide range of apoB-containing lipoproteins (whichever is available, as long as the receptor-binding region is accessible). The effect of PCSK9 inhibition on triglycerides has been discussed in the past, generally showing little effect on plasma triglyceride levels. But more recent metabolic studies indicate that PCSK9 inhibitors may enhance the clearance of triglyceride rich postprandial lipoproteins in obesity [32, 33]. This is consistent with the data shown here.

A limitation of the study represents the small number of subjects evaluated. Thus, we could not study the wide range of lipid abnormalities (normal lipid status, hypertriglyceridemia, hypercholesterolemia, mixed hyperlipidemia) shown in FDBL patients. In our study, we evaluated patients with the typical dyslipidemia and therefore our conclusions are also limited to this patient subgroup. On the other hand, it is very likely that a similar result can also be expected in patients with FDBL presenting with other lipid phenotypes. Another limitation is that we only studied the effect of evolocumab, but again it is likely that results would be identical with other PCSK9-targeting therapies.

Taken together, our results indicate that PCSK9-inhibition reduces the concentration of a wide variety of apoB-containing lipoproteins with the dominant particle size being reduced the most. Thus, in LDL hypercholesterolemia or mixed hyperlipidemia where most apoB-containing lipoproteins are LDL, the LDL fraction is reduced while the concentration of a broader range of lipoproteins is reduced in FDBL. It can be safely assumed that this reduction in apoB-containing lipoproteins translates into clinical benefit.

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29 Nov 2021

Effect of PCSK9 inhibition on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)

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Reviewer #1: Although I believe the study is interesting, it contains some issues that need to be sorted out.

The introduction misses the part relative to PCSK9, e.g., the role of PCSK9 on the expression of LDL-R, and the general impact of PCSK9 inhibition in terms of LDL-C lowering and atherosclerotic cardiovascualr disease (ASCVD) risk reduction. Concerning the biology of PCSK9, the Authors can refer to a recent review (PMID: 34019847). A similar comment is valid in the case of lipoprotein(a). Relative to this, the discussion lacks to mention the impact of PCSK9 inhibition on lipoprotein(a). Moreover, lipoprotein(a) levels seem to be very high and in the hypercholesterolemic group, they overtake the ASCVD risk threshold of 50 mg/dL. The Authors should evaluate the contribution of cholesterol carried by lipoprotein(a), and calculate, in addition, the corrected LDL-C (PMID: 34450317). Considering that, for lipoprotein(a), conversion between mg/dL and mmol/L is not advisable, cholesterol should be expressed both in mmol/L and mg/dL. Please report the assay that has been used to measure lipoprotein(a).

Relative to ultracentrifugation, is it correct “48 hours at 158°C?”

Which statistical test has been used to evaluate differneces? My apology if I missed it.

The impact of PCSK9 on triglycerides should be discussed in more details. Please refer to the review by Dijk W et al (PMID: 29665987) or Baragetti et al (PMID: 29428206).

Which is the percentage of recurrence of FDBL? This could be reported both in the introduction and in the limitation section. Please report that it is a single centre study. The title should report that data have been obtained with evolocumab.

Reviewer #2: In this study, the Authors investigated the impact of PCSK9 inhibition on lipoproteins in patients with FDBL. A comparison with patients with LDL-hypercholesterolemia and mixed hyperlipidemia has been made. PCSK9 inhibitors reduced cholesterol and apoB in all patients. In FDBL they specifically reduced VLDL-cholesterol. The Authors concluded that PCSK9-inhibition reduces the concentration of a wide variety of apoB-containing lipoproteins with the dominant particle size being reduced the most.

The study is original and interesting, however the paper is only descriptive, since sample size is too small and statistical analysis could not be performed.

This reviewer is aware of the rareness of familial dysbetalipoproteinemia, however three patients are not so much to draw robust conclusions, mostly because of the huge variability in lipid and lipoprotein profile.

Thus, more efforts should be dedicated to enroll additional subjects and increase sample size.

Other comments:

Tables: how are data reported? Please provide whether the number are mean ± SD or median ± SEM.

Table 1: which is the prevalence of metabolic syndrome in these patients.

Figure 2 is confusing. I would suggest using 3 different panels.

Non si reduce solo il picco più alto. Si riduce tutto in proporzione

State which inhibitor has been used also in method section, since it is reported only in the abstract.

Reviewer #3: The study is interesting but some consideration has to be done.

Please consider revising the description of the disease.

Aim. From your introduction, I do not understand what you want to test by comparing these population. Which differences do you expect? Why this study is relevant?

LDL-hypercholesterolemia, I imagine that these patients are heterozygous FH. Please use the correct terminology.

I do not understand the population selection:

1) Please do not give in methods any information on the demographic or clinical characteristics of these patients. These are reported in Table 1.

2)Did you genotype your patients? Both ApoE2/E2 patients and HeFH have already received a molecular confirmation? If yes, was it done for scientific purpose for this study or for clinical reasons?

3)Nevertheless, how did you choose patients with mixed dyslipidemia and HeFH to be compared with FDBL. Did you take all your patients taking PCKS9i?

“All had the indication for treatment with PCSK9-inhibitors according to German

regulatory standards (not reaching treatment goals recommended by the European guidelines despite

maximally tolerated oral lipid-lowering therapy)” please give a reference otherwise it is difficult to understand patient’s characteristics.

Where PCKS9i given in a study protocol? I do not understand the study design. I guess if it is an observational real-world study or an interventional trial.

As you assess only lipoprotein fraction at baseline and 3 months a suggest you consider in your discussion that your results might be in “acute” phase of treatment.

Add significance to Table 1 (BMI is significantly different between groups)

Please define CVD, CAD and PVD. Did you consider only acute events or all the events?

Please also give statistics and the methodology used to analyze the data (reporting it in a separate chapter.

Inferential statistics is completely lacking. You should add it throughout the manuscript (including Table 2 and figures)

Moreover, there was any changes in the background lipid lowering therapies in the 3 months of treatment? Please add lipid lowering therapies to table 2.

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Reviewer #2: No

Reviewer #3: No

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21 Dec 2021

We thank the Editor and the reviewers for their thoughtful and constructive comments. We have incorporated them into the revised version and believe that this has considerably improved the manuscript. For details look at the upöoaded documents (Response to Reviewers; Cover letter R1). We hope that the manuscript will now be fond acceptable for publication.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Laura Calabresi

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: N/A

Reviewer #3: N/A

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Almost all comments have been addressed. Sample size remains a major issue that cannot be resolved.

Minor comments:

Please revise abbreviations and typos (e.g. Celcius, PCSK-9 or PCSK9)

Reviewer #3: Dear author my major concearn is the lack of an accurate definition of the patients included in the study.

In fact, I am awared that familial dysbetalipoproteinemia is rare but for this reason, patients should be very well characterized. Similarly, the "control" population lack of definition as for example, you have included patients with FH with those suffering with other form of hypercholesterolemia in a unique group. I believe that these could be potentially confounders. I suggest publishing this data as a short communication rather than as an original article.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Feb 2022

Comments were addressed in the revised version of the manuscript. Please see "Cover letter" and "Response to reviewers".

Regards

Klaus Parhofer

Submitted filename: Response to Reviewers R2.pdf

Click here for additional data file.

9 Mar 2022

Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)

PONE-D-21-26920R2

Dear Dr. Parhofer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Laura Calabresi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: Thank you for your replay and having added further information on patients. I still believe that molecular diagnosis could have added some further interesting to this observation.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

15 Mar 2022

PONE-D-21-26920R2

Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia)

Dear Dr. Parhofer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Laura Calabresi

Academic Editor

PLOS ONE