Mutations in LPL, APOC2, APOA5, GPIHBP1 and LMF1 in patients with severe hypertriglyceridaemia

Abstract

Objective

The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG >10 mmol/L) it is a challenge to define the underlying cause. We investigated the molecular basis of severe HTG in patients referred to the Lipid Clinic at the Academic Medical Center Amsterdam.

Methods

The coding regions of LPL, APOC2, APOA5 and two novel genes, lipase maturation factor 1 (LMF1) and GPI-anchored HDL-binding protein 1 (GPIHBP1), were sequenced in 86 patients with type 1 and type 5 HTG and 327 controls.

Results

In 46 patients (54%) rare DNA sequence variants were identified, comprising variants in LPL (n=19), APOC2 (n=1), APOA5 (n=2), GPIHBP1 (n=3) and LMF1 (n=8). In 22 patients (26%) only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) could be identified, whereas no mutations were found in 18 patients (21%). In vitro validation revealed that the mutations in LMF1 were not associated with compromised LPL function. Consistent with this, five of the eight LMF1 variants were also found in controls and therefore cannot account for the observed phenotype.

Conclusion

The prevalence of mutations in LPL was 34% and mostly restricted to patients with type 1 HTG. Mutations in GPIHBP1 (n=3), APOC2 (n=1) and APOA5 (n=2) were rare but the associated clinical phenotype was severe. Routine sequencing of candidate genes in severe HTG has improved our understanding of the molecular basis of this phenotype associated with acute pancreatitis, and may help to guide future individualized therapeutic strategies.

Introduction

Severe hypertriglyceridaemia (HTG) is characterized by plasma triglyceride (TG) levels >10.0 mmol/L [1]. Clinical features in patients with severe HTG often include recurrent pancreatitis, eruptive xanthomas and lipaemia retinalis [2, 3]. Both genetic and lifestyle factors contribute to elevated plasma TG concentrations although the genetic component remains largely undefined [4].

Lipoprotein lipase (LPL) deficiency is a major cause of severe HTG. LPL has the unique ability to hydrolyse circulating TGs which are packaged in chylomicrons (dietary lipids) or very-low density lipoproteins (VLDL; lipids of hepatic origin). LPL is present at the cell surface of endothelial cells in small capillaries of tissues requiring fatty acids for their source of energy (heart and skeletal muscle) or for storage (adipose tissue). A complete or near-complete absence of catalytically active LPL invariably leads to severe HTG.

HTG is a hallmark of both Fredrickson type 1 and type 5 hyperlipidaemias. Type 5 hyperlipidaemia encompasses a mixed hyperlipidaemic phenotype including elevated levels of both chylomicron and VLDL remnant particles [5]. The molecular basis of type 5 HTG has not been fully elucidated, but often include heterozygosity for a loss of function mutation in LPL as a background. Congenital LPL deficiency (type 1 hyperlipidaemia or hyperchylomicronaemia syndrome) is a rare disorder with an estimated prevalence of one in half a million in the general population. Type 1 HTG is a monogenic disorder frequently caused by loss of function mutations in LPL. It is interesting that mutations in APOC2 encoding apolipoprotein (apo) C-II, which is an essential cofactor for LPL activity, and in APOA5 encoding apo A-V, which is a modulator of LPL function, have been reported in patients with severe HTG [6, 7]. Recently, two new proteins were identified that were shown to be essential for proper LPL function: lipase maturation factor 1 (LMF1) and glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1).

LMF1 has been shown to be essential for the maturation of both LPL and hepatic lipase (HL) to their fully functional forms [8]. Of note, two homozygous nonsense mutations in LMF1 were recently identified in two patients with severe HTG leading to combined lipase deficiency [8, 9].

GPIHBP1 has been identified as the endothelial protein that facilitates LPL trafficking towards the endothelial cell surface and provides a platform for TG lipolysis [10, 11]. Homozygous mutations in GPIHBP1 abolish LPL binding to GPIHBP1 and thus impair TG lipolysis. To date, seven mutations and one large deletion in GPIHBP1 have been reported in patients with severe HTG [12–19]. A putative GPIHBP1 binding site in LPL has been identified and lies downstream of the heparin-binding site between amino acids 443 and 462. These data provide an explanation for the severe HTG phenotype in patients with a missense mutation in this region of LPL [20].

Because therapeutic interventions aimed at lowering TG levels in patients with severe HTG are often ineffective and might partially depend upon the exact molecular pathophysiology, insight into the molecular basis of severe HTG may guide individualized therapeutic strategies. In the present study we set out to define the molecular and clinical abnormalities in 86 patients with severe HTG (both type 1 and type 5) who presented at a tertiary referral centre. The coding regions of LPL, APOC2, APOA5, GPIHBP1 and LMF1 were sequenced.

Methods

Study participants

A total of 86 patients, fulfilling the criteria of severe HTG (TG >10 mmol/L) and referred to the Lipid Clinic at the Academic Medical Center Amsterdam, were included in the present study. Forty-three patients were identified as having type 1 HTG with post-heparin LPL activity ≤ 30% of the level measured in a pooled control sample. Exclusion criteria were APOE2/E2 genotype, alcohol abuse and prolonged uncontrolled diabetes (HbA_1C >8.5%). Additionally, 327 population-based controls were included in the study [21]. Written informed consent was obtained from all participants.

Lipid analysis and post-heparin LPL activity

Blood samples were drawn, after an overnight fast, into EDTA-coated tubes for lipid and apolipoprotein analysis. Post-heparin blood was collected in heparin-coated tubes 15 min after an intravenous heparin bolus (50 IU/kg bodyweight, Leo, Breda, The Netherlands) [13]. Blood was stored on ice directly after withdrawal. Plasma was isolated by centrifugation at 3000 rpm at 4°C for 15 min and stored in aliquots at −80°C until required for further analyses. Total plasma cholesterol, TG, high-density lipoprotein cholesterol (HDLc) and low-density lipoprotein cholesterol (LDLc) levels were determined with commercial kits (Wako, Japan). Plasma apo B, apo C-II and apo C-III levels were measured with commercial assays (Randox, USA). All analyses were performed on a Cobas Mira autoanalyser (Roche, Basel, Switzerland). LPL mass was measured using a commercially available kit (Markit-M LPL, Dainippon Pharmaceutical Co, Osaka, Japan). LPL and HL activity were analysed as described previously [13]. In short, lipase activity assays were performed using gum acacia-stabilized (³H)-trioleylglycerol as a substrate. HL activity was determined after inhibition of LPL for 2 h at 4°C with a mouse monoclonal antibody directed against human LPL (5D2, a generous gift from J.D. Brunzell, MD PhD, Seattle, WA, USA). Fatty acids were extracted as calcium salts and radioactivity was counted in a liquid scintillation counter. LPL activity was calculated by subtracting total lipase activity from HL activity. Lipase activities are expressed as percentage of a control post-heparin ‘pool’ obtained from post-heparin plasma from 20 healthy individuals.

Analysis of candidate genes

Genomic DNA was extracted from 10 mL whole blood on an AutopureLS apparatus according to the manufacturer’s instructions (Gentra Systems, Minneapolis, MN, USA). For sequence reactions, primer pairs were designed using Primer3 software to cover all coding regions and intron–exon boundaries of LPL (NM_000237.2), LMF1 (NM_022773.2), GPIHBP1 (NM_178172.3), APOC2 (NM_000483.3) and APOA5 (NM_052968.3) (Supplementary Table 1). An M13 tail was added to each primer (forward: 5′-GTTGTAAAACGACGGCCACT-3′; reverse: 5′-CACAGGAAACAGCTATGACC-3′) to facilitate DNA sequencing. Sequence reactions were performed as described[12,13]. Nucleotides were numbered according to the official nomenclature provided by the Human Genome Variation Society (www.hgvs.org/mutnomen). The coding DNA numbering starts at the ATG transcription start site and ends with the stop codon. This results in differences in LPL mutation nomenclature, i.e. p.S447X is now termed p.S474Ter. APOE genotyping was performed using allelic discrimination with pre-designed primers (Applied Biosystems, Foster City, CA, USA). PCR was performed according to the manufacturer’s instructions in a CFX96 PCR apparatus (BioRad, Hercules, USA, CA, USA) using Tagman assay mix (Applied Biosystems). In the control population, sequencing was performed as previously described [22].

All variants were checked in the dbSNP123 and 1000-genome databases for their rs numbers. Common variants had an minor allele frequency (MAF) >0.05. Mutations were defined as missense, nonsense or frameshift changes in genomic DNA sequences. For all mutations, in silico analyses were performed with the PolyPhen and SIFT programs to predict the functional consequences of the observed amino acid substitutions.

Functional analysis of LMF1 mutations

Functional analysis of LMF1 was performed as described previously [23]. In brief, human LMF1 cDNA including a c-myc N-terminal epitope tag was subcloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA). Mutations were introduced by site-directed mutagenesis (Stratagene, Santa Clara, USA, CA, USA). The expression vectors were co-transfected with a plasmid encoding human LPL into a homozygous cld mutant (i.e. Lmf1-deficient) mouse hepatocyte cell line. A secreted human placental alkaline-phosphatase (SEAP) reporter gene subcloned into the pM1 expression vector (X-extremeGENE, Roche, Switzerland^®) was used to normalize for transcription efficiency. An LPL/SEAP plasmid master mix was prepared and used in all assays. LMF1 promotes maturation of the LPL protein and is thus essential for activation of the protein [8]. Therefore LMF1 function was assessed by measuring the rate of secretion of active LPL into the cell culture medium normalized by SEAP activity. Two LMF1 mutants, p.Tyr439Ter [8] and p.Trp464Ter [9] which have previously been shown to affect LMF1 function, were used as controls. All data were normalized with respect to the results of wild-type LMF1 protein.

Statistical analysis

All data are presented as mean ± standard deviation. Differences in gene frequencies between cases and controls were tested using chi-squared statistics. Differences in LPL secretion was evaluated by analysis of variance and the Student’s t test. A P-value <0.05 was considered statistically significant.

Results

Patient characteristics and lipid and apolipoprotein measurements are summarized in Table 1. We included 43 patients with type 1 and 43 patients with type 5 hyperlipidaemia. Plasma TG was above 10 mmol/L in all patients. By definition, type 1 patients had strongly impaired plasma post-heparin LPL activity levels (<30% of control). Plasma apo B levels were higher in patients with type 5 than in those with type 1 HTG, reflecting the presence of both chylomicron and VLDL remnant particles. Increased levels of plasma apo C-II and apo C-III were measured in both groups. In total, 41% of patients (20 type 1 and 15 type 5 HTG) had a history of pancreatitis.

Table 1.

Characteristics of 86 patients with severe HTG

Characteristic	Type 1	Type 5
Male/female, n	30/13	29/14
Age, years	37 (1–69)	45 (17–69)
BMI, kg/m2	26 (20–35)	28 (20–42)
ApoE genotype, n
E3E3	19	15
E2E3	6	10
E4E3	8	8
E4E4	1	3
E2E4	1	3
Pancreatitis, n	20	15
Triglycerides, mmol/L	41.5 (11.5–175.0)	32.0 (10.1–128.1)*
Cholesterol, mmol/L	6.5 (1.3–15.3)	5.6 (3.1–15.0)
HDL cholesterol, mmol/L	0.7 (0.1–1.9)	0.8 (0.1–1.7)
LDL cholesterol, mmol/L	2.3 (0.6–6.1)	2.7 (0.4–5.3)
Apolipoprotein B, mg/dL	75 (23–137)	100 (50–163)**
Apolipoprotein A-I, mg/dL	109 (61–190)	130 (64–184)**
Apolipoprotein C-II, mg/dL	19.0 (0.2–48.8)	13.2 (1.5–25.9)
Apolipoprotein C-III, mg/dL	37.3 (10.1–98.7)	23.0 (6.4–40.1)
LPL activity, (%)	5.3 (0–27)	87 (32–>100)*

Data are presented as mean (minimum–maximum). Triglycerides are presented as median (minimum–maximum) based on the highest plasma triglyceride levels recorded. LPL activity is presented as percentage of control pool. Data were available for ApoE genotype, Chol, HDLc, LDLc, apoB, apo A-I, apo C-II, apo C-III and LPL activity for 74, 66, 59, 48, 57, 59, 35, 36 and 75 individuals, respectively. Differences in baseline characteristics were tested for significance using the Kruskal–Wallis test.

^*P<0.005;

^**P<0.05.

Genetic analyses of candidate genes

We identified 18 known mutations (14 in LPL, one in APOC2, one in APOA5 and two in GPIHBP1) and 16 novel mutations (five in LPL, one in APOA5, two in GPIHBP1 and eight in LMF1) in 46 patients (53%) (Table 2). In 22 patients (26%) only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) were found. No sequence variants could be identified in the remaining 18 patients (21%).

Table 2.

Genetic variants identified in candidate genes in patients with severe HTG.

Gene	Exon	Variation	Position	Polyphen/SIFT prediction	Loss of function	Ref	Hom	Htz	Freqency in controls ** (n, %)
LPL	2	p.Asp36Asn (rs1801177)*	c.106G>A	Possibly damaging/tolerated	Yes	[36	0	6 (7%)	9 (2.8%)a
	3	p.Val96Leu	c.286G>C	Probably damaging/tolerated	Yes	[37	0	4	-
	4	p.Gly161Glu	c.482G>A	Probably damaging/deleterious		-	1	0	-
		p.Gly166Ser	c.496G>A	Probably damaging/tolerated	Yes	[38	1	0	-
		p.Gly181Ser	c.541G>A	Probably damaging/deleterious	Yes	[39	3	0	-
		p.Asp183Gly (rs118204064)	c.548A>G	Probably damaging/deleterious	Yes	[40	5	0	-
	5	p.Pro184Arg	c.551C>G	Probably damaging/deleterious	Yes	[41	1	0	-
		p.Arg197His	c.590G>A	Probably damaging/tolerated		[27	0	1	-
		p.Val206Ala	c.617T>C	Probably damaging/deleterious		-	0	1	-
		p.Gly215Glu (rs118204057)	c.644G>A	Possibly damaging/deleterious	Yes	[42	0	4	-
		p.Ile252Thr (rs118204080)	c.755T>C	Probably damaging/deleterious	Yes	[43	0	1	-
	6	p.Arg270Cys (rs118204077)	c.808C>T	Probably damaging/deleterious	Yes	[44	0	1	-
		p.Arg270His (rs118204062)	c.809G>A	Probably damaging/deleterious	Yes	[43	0	1	-
		p.Asp277Asn (rs118204068)	c.829G>A	Probably damaging/deleterious	Yes	[45	0	1	-
		p.Ser278Cys	c.833C>G	Probably damaging/deleterious	Yes	[38	0	1	-
		p.Leu301SerfsX3	c.901del		Yes	-	0	1	-
		p.Asn318Ser (rs268)*	c.953A>G	Benign/tolerated	Yes	[36	2	10	4 (1.2%)c
	7	p.His348GlnfsX43	c.1044_1050del		Yes	-	0	1	-
		p.Leu380AlafsX2	c.1138_1139del		Yes	[46	2	0	-
		p.Thr379Ile (rs76708715)	c.1136C>T	Benign/tolerated		[47	0	1	-
	8	p.Ala427Thr (rs5934)	c.1279G>A	Benign/tolerated		-	0	3	-
	9	p.Ser474Ter (rs328)*	c.1421C>G		Gain	[36	2	6	42(12.8%)d
APOC2	2	p.Val40Ter	c.118del		Yes	[6]	2	1	-
APOA5	2	p.Ser19Trp (rs3135506)	c.56C>G	Possibly damaging/deleterious			2	16	30 (9.2%)c
		p.Gly271Cys	c.811G>T	Benign/deleterious		[25	0	1	-
	3	p.Thr133Arg	c.398C>G	Probably damaging/deleterious		-	0	1	-
GPIHBP1	3	p.Cys65Tyr	c.194G>A	Probably damaging/deleterious	Yes	[13	1	0	-
	4	p.Thr108Arg	c.323C>G	Possibly damaging/tolerated		-	1	0	-
		p.Gln115Pro	c.344A>C	Probably damaging/deleterious	Yes	[12	1	0	-
		p.Ser144Phe (rs783672430	c.431C>T	Possibly damaging/deleterious		-	0	1	-
LMF1	1	p.Gly36Asp (rs111980103)	c.107G>A	Benign/tolerated		-	0	7 (8%)	65
	5	p.Arg230Gln	c.689G>A	Benign/tolerated	No	-	0	1	-
	6	p.Arg264Cys	c.790C>T	Probably damaging/deleterious	No	-	0	2	-
	7	p.Arg351Gln	c.1052G>A	Benign/tolerated	No	-	0	2 (2.3)	2 (0.6%)d
		p.Arg354Trp	c.1060C>T	Possibly damaging/deleterious	No	-	0	3 (3.5)	13 (4.0%)d
	8	p.Arg364Gln (rs35168378)	c.1092G>A	Probably damaging/deleterious	No	-	0	3 (3.5)	13 (4.0%)d
	11	p.Arg523His	c.1568G>A	Possibly damaging/tolerated	No	-	1	0	-
		p.Pro562Arg (rs4984948)	c.1685C>G	Probably damaging/deleterious	No	-	0	1 (1%)	3 (0.9%)d

^*Old nomenclature: p.Asp9Asn, p.Asn291Ser and p.Ser447X.

^**Rare variants in LPL, APOC2, APOA5, GPIHBP1 were not present in controls (-).

^***Differences were statistically significant using chi-squared statistics with

^aP<0.05;

^bP<0.02;

^cP<0.001;

^dnot significantly different. Htz: heterozygous, Hom: homozygous.

LPL

Sequencing LPL provided 14 different known mutations in 22 patients (Table 2). Thirteen patients were carriers of a homozygous mutation in LPL, four patients had a compound heterozygous mutation and five carried a single heterozygous mutation. Among the five novel mutations in LPL, the p.Gly161Glu and p.Val206Ala mutations were predicted to be ‘probably damaging’ by PolyPhen and ‘deleterious’ according to SIFT, whereas the p.Ala427Thr mutation (rs5934) was predicted to be ‘benign’ and ‘tolerated’, respectively. Two frame shift mutations, LPL p.Leu301SersfX3 and p.His348GlnfsX43, caused a premature stop. Characteristics of the carriers of the novel sequence variants are shown in Table 3. In line with the in silico predictions, the carriers of the p.Leu301SersfX3 and p.Gly161Glu mutations had strongly reduced post-heparin LPL activity with the characteristics of type 1 HTG. Measurement of LPL activity was not available for the patient carrying the p.His348GlnfsX43 mutation. Carriers of the p.Val206Ala and p.Ala427Thr mutations had normal post-heparin plasma LPL activity levels. Data from two additional carriers of the p.Ala427Thr mutation were incomplete and are therefore not presented in Table 3. None of the novel mutations was present in the control population. In supplementary Table 2 the number of individual mutations in LPL in patients with type 1 and type 5 HTG is provided.

Table 3.

Novel missense and nonsense variants in LPL, APOA5 and GPIHBP1 and biochemical characteristics of each patient

Gene	Mutation		Second mutation		Gender	Age (years)	BMI (kg/m2)	Pancreatitis	Max TG mmol/L	LPL mass ng/mL	LPL activity % **
LPL	p.Gly161Glu	Hom	LPL: p.Asn318Ser	Htz	M	39	23	Yes	19.2	223	60
	p.Val206Ala	Htz			F	53	20	No	34.6	Na	>100
	p.Leu301SerfsX3	Hom	LPL: p.Asn318Ser	Htz	F	39	31	No	22.3	Na	23*
	p.His348ProfsX43	Htz			M	52	23	No	17.6	Na	Na*
	p.Ala427Thr	Htz	LMF1:p.Gly36Asp	Htz	F	51	30	No	90.8	132	>100
APOA5	p.Thr133Arg	Htz			M	49	27	Yes	85.3	65	0*
GPIHBP1	p.Thr108Arg	Hom			M	1	Na	Yes	Na	Na	Na
	p.Ser144Phe	Htz			F	45	45	Yes	35.8	10	>100
LMF1	p.Arg230Gln	Htz	p.Arg523His	Hom	M	31	23	No	51.8	Na	>100
	p.Arg264Cys	Htz	p.Gly36Asp	htz	M	40	29	Yes	15.4	na	79

^*Type 1 HTG phenotype.

^**LPL activity is given as percentage of plasma pool. Htz: heterozygous, Hom: homozygous, M: male, F: female, Na: not available. Max TG: maximal plasma TG levels. BMI, body mass index.

Finally, three common coding variants in LPL were found, including p.Asp36Asn (rs1801177, n=6), p.Asn318Ser (rs268, n=12) and p.Ser474Ter (rs328, n=8) (Table 2). The p.Asp36Asn and p.Asn318Ser variants were significantly more common in HTG patients than in controls. By contrast, the p.Ser474Ter variant was found at a higher frequency in the controls, which is in line with the proposed gain of function of this variant.

APOC2 and APOA5

Three family members were found to be homozygous for a nonsense mutation in APOC2 (p.Val40Ter) resulting in a concomitant absence of apo C-II protein. The complete characterization of the mutation has been described in detail previously [6, 24]. We identified one patient with a heterozygous mutation in APOA5 (p.Gly271Cys), which has been discussed previously [25]. In addition, a novel heterozygous mutation in APOA5 (p.Thr133Arg) was found in a patient with normal post-heparin plasma LPL mass but no post-heparin plasma LPL activity and a history of pancreatitis. The variant was predicted by PolyPhen and SIFT to be ‘probably damaging’ and ‘deleterious’, respectively.

The common variant in APOA5, p.Ser19Trp (rs3135506), was found in 18 patients in our cohort, including two homozygous carriers (Table 2). The observed frequency of this common variant was significantly higher in the patient group compared to controls (21% vs. 9%).

GPIHBP1

Two homozygous missense mutations in GPIHBP1 (p.Gln115Pro and p.Cys65Tyr) were identified; both result in a complete absence of binding of LPL to GPIHBP1 [12, 13]. A novel homozygous mutation (p.Thr108Arg) was identified in a 1-year-old child with a history of pancreatitis (Table 2). This mutation is located adjacent to a cysteine residue at 110. Plasma LPL activity measurements were not available for this very young child. On a very restricted formula diet, the plasma TG concentration decreased to 4.3 mmol/L. This mutation was predicted by Polyphen and SIFT to be ‘possibly damaging’ and ’tolerated’, respectively.

A heterozygous missense mutation in GPIHBP1, p.Ser144Phe (rs78367243), was found in an HTG patient with normal post-heparin plasma LPL activity levels, but low LPL mass. These clinical characteristics are consistent with the lack of homozygosity and thus lack of complete loss of function. The in silico prediction programs predict ‘possibly damaging’ (Polyphen) and ‘deleterious’ (SIFT). None of these variants was found in controls.

LMF1

Eight novel missense variants in LMF1, including one homozygous mutation (p.Arg523His), were found in 15 patients (Table 2). The variants p.Arg264Cys, p.Arg354Trp, p.Arg364Gln, p.Arg523His and p.Pro562Arg were predicted to be ‘probably damaging’ by PolyPhen and ‘deleterious’ by SIFT. Functionality was assessed for all LMF1 variants except the p.Gly36Asp. LMF1 variants were expressed in cld-mutant hepatocytes in combination with an LPL expression vector. None of the LMF1 variants led to a reduced LPL activity in the cell medium. By contrast, two previously characterized nonsense mutations (p.Tyr439Ter and p.Tyr464Ter) significantly abolished LPL activity in the medium (Figure 1). Through searching in the most recent dbSNP123 database it was clear that three LMF1 variants have been recently described and have been given an rs number (Table 2). LMF1 sequence analysis revealed that the p.Gly36Asp (rs111980103) variant occurred with a higher frequency (19.9%) in controls than in HTG patients (8.0%; P<0.02). The p.Arg354Trp and p.Arg364Gln variants occur at similar frequency in cases and controls (3.5% vs. 4.0%). In single LMF1 mutation carriers, plasma post-heparin LPL levels were normal or only moderately reduced and therefore cannot account for the severe HTG phenotype (Table 3).

Figure 1. Functional analysis of LMF1 mutants.

LMF1 function was tested in vitro in cld-mutant hepatocytes co-expressing LPL and LMF1. Wild-type LMF1 protein shows release of LPL activity in the medium. This activity level was set as 1. LMF1-W464X and LMF1-Y439X result in loss of LMF1 function and thus severely impaired release of active LPL. All newly diagnosed LMF1 variants show a normal release of active LPL and thus do not affect LMF1 function. Means ± SE are shown; *P<0.05.

Figure 2 shows an evaluation of the data according to type 1 and type 5 phenotype. Among patients with type 1 HTG, 51% had a mutation in LPL, including all patients with homozygous mutations. Only 16% of patients with type 1 HTG had a loss of function mutation in APOC2, GPIHBP1 or APOA5. Additionally, 21% of the type 1 patients were carriers of only a common single-nucleotide polymorphism (SNP) in LPL or APOA5, whereas no genetic aberration could be found in 12% of patients. In these patients the causal mutation has yet to be identified. In patients with type 5 HTG, the occurrence of genetic variations in LPL only accounted for 19% of cases. Carriers of only common SNPs in LPL and APOA5 were found more frequently in type 5 patients (37%) whereas no genetic variations in the candidate genes were identified in 37% of patients. Thus, the genetic background in patients with type 5 HTG will require additional sequencing efforts to identify the causal mutations responsible for the HTG phenotype.

Figure 2. The distribution of genetic variants in candidate genes for severe HTG type 1 and type 5.

Among patients with type 1 HTG, 51% carry a mutation in LPL (13 have a homozygous mutation, two are compound heterozygote, four are heterozygous and three are carriers of a novel mutation). Seven type 1 HTG patients (16%) are carriers of a rare mutation in APOC2, APOA5 and GPIHBP1. Nine patients carry a common variant in LPL or APOA5 and five patients did not have any mutation in the candidate genes.

Among type 5 patients, only 19% were carriers of a mutation in LPL (two compound heterozygous, three heterozygous for one mutation and three were carriers of a novel mutation). Only one carrier was identified with a heterozygous mutation in APOA5. There were two carriers with a mutation in LMF1. Sixteen patients (37%) had a common genetic variation in LPL or APOA5 and 17 patients (40%) did not have any genetic abnormality in the candidate genes.

Discussion

We identified rare genomic variants in 46 (54%) patients with severe HTG through sequencing five candidate genes known to be involved in LPL function. In total, 19 known and 16 novel disease-causing mutations were identified in LPL, APOC2, APOA5 and GPIHBP1. In 29 patients (34%), a mutation in LPL was the sole underlying cause of HTG. Mutations in APOC2, APOA5 and GPIHBP1 were rare and were cumulatively found in only 11% of the patients. The eight novel mutations in LMF1 were, however, not associated with reduced LPL activity. These findings imply that DNA screening in cohorts with severe HTG, at least in lipid referral centres, is an efficient strategy to unravel the molecular diagnosis of HTG which may help to guide future individualized therapeutic strategies.

The prevalence of LPL variants in our cohort was high compared to previous observations. Wang et al. sequenced LPL, APOC2 and APOAV in 110 non-diabetic subjects with severe HTG [26]. They reported a heterozygous mutation in LPL or APOC2 in 10% of patients. Wright et al. identified mutations in LPL in eight of a total of 19 (42%) patients with severe HTG. The majority of patients, however, were carriers of the frequent missense mutations p.Asp36Asn and p.Asn318Ser [27]. In a recent study in 107 German patients with plasma TG levels >10 mmol/L, 13 patients were identified with a loss of function mutation in LPL (12%), which is substantially lower than observed in our cohort, but may be a consequence of less stringent inclusion criteria [28]. It is interesting that only three of the rare mutations identified in the German cohort were found in our HTG patients. In the present study, we found rare mutations in LPL in 30 (35%) patients after exclusion of the frequent variations. The relatively high prevalence of rare mutations in our cohort may be due to ‘selection’ bias, as patients with the most severe phenotype are often referred to a tertiary lipid clinic.

Most of the LPL variants in our cohort were characterized as loss of function mutations with documented functional impairment. Based upon both bioinformatic analysis and a significant decrease in post-heparin LPL activity observed in the mutation carrier, it has been proposed that several novel variants are functional. The p.Gly161Glu and p.Val206Ala mutations are located in exon 4 and exon 5, respectively, within the catalytic triad where the sequence is highly conserved across multiple species. These mutations are thus predicted to lead to loss of function of the protein. However, both mutations occur in a heterozygous state. The p.Ala427Thr is located in exon 8 and this sequence is not highly conserved among species. This explains the weak effect of the mutation on the observed phenotype in patients. A substantial number of type 5 HTG patients in our cohort were carriers of mutations in LPL that lead to only a small elevation of plasma TG level. However, in the presence of exogenous challenges (high-fat diet, obesity, diabetes, alcohol abuse), or when additional common genomic variants are present, milder variants of LPL may also result in more severely disturbed TG metabolism.

The p.Ser474Ter was found in eight patients (9%), which is lower than in the controls and in keeping with observations in other HTG cohorts [26, 29]. The p.Ser474Ter has proven to be a gain of function mutation with therapeutic potential as shown in a recent gene-therapy trial in patients with LPL deficiency [30]. However our observations suggest that the p.Ser474Ter variant is not ‘protective’ in patients with severe HTG.

Variants in APOC2, APOA5 and GPIHBPI were rare. One mutation in APOC2 was found, leading to a premature stop codon [6]. Because apo C-II is an essential co-factor for LPL to exhibit its activity in plasma, deficiency of apo C-II protein ultimately leads to a severe LPL deficiency phenotype. We identified only one known and one novel heterozygous mutation in APOA5 (p.Thr133Arg). The variant was predicted to be ‘likely functional’ by PolyPhen. In line with this, the patient with this novel variant had reduced post-heparin plasma LPL activity. The severity of the phenotype and the identification of a heterozygous mutation in APOA5 suggests that the presence of another mutation in a yet unknown gene might contribute to the observed phenotype. To address this issue we will sequence the complete exome. Of note, the exact role of APOA5 mutations in HTG remains controversial [7]. It has been postulated that rare variants in APOA5 only impact on TG levels if present in a homozygous form or present with concomitant common APOA5 variants [25]. The common variant in APOA5, p.Ser19Trp (rs3135506), was found in 18 (21%) patients, which is 2-fold higher than the frequency reported in other populations but is consistent with previous findings of elevated allele frequencies for this common variant in HTG [26, 31, 32].

We identified three homozygous mutations and one heterozygous mutation in GPIHBP1. All variants were located in the Ly-6 domain which is the essential part of the protein for LPL binding [33]. In cell culture experiments, this mutated form of GPIHBP1 reached the cell surface but lacked the ability to bind LPL [12–14]. In an earlier study we observed that heterozygosity for a loss of function mutation in GPIHBP1 (p.Cys65Tyr) did not affect plasma TG levels, implying that heterozygosity for a mutation in GPIHBP1 may not be enough to cause the HTG phenotype [13]. Furthermore, the heterozygous p.Ser144Phe carrier had normal LPL activity and mass.

In the present HTG cohort we sequenced the recently identified LMF1 gene. Eight novel variants were found in 15 patients. LMF1 is a membrane-bound protein localized in the endoplasmic reticulum and is essential for the maturation of both LPL and HL to their functional forms [8]. Thus, loss of function mutations in LMF1 are expected to affect LPL activity. In line with this concept, two homozygous nonsense mutations in LMF1 were independently described in two individual patients with severe chylomicronaemia [8, 9]. Whereas some of the variants identified in our cohort were predicted to be ‘likely functional’ by PolyPhen, no abnormal plasma post-heparin LPL activities were measured in LMF1 mutation carriers. Furthermore, in vitro testing revealed that none of the mutations in LMF1 abolished the activity of secreted LPL. Collectively, these data indicate that LMF1 function may only be impaired in the presence of a homozygous nonsense mutation that completely disrupts the functional unit of the protein.

The number of patients without any genetic abnormalities or only common variants in the candidate genes was higher in type 5 compared to type 1 HTG patients. These findings are consistent with the definition of type 1 hyperlipidaemia. Whereas type 1 hyperlipidaemia is believed to be a monogenetic disorder, type 5 hyperlipidaemia has recently been suggested to be a more complex polygenetic trait including genetic determinants of more modest variation in TG levels [21].

Study limitations

Several limitations of the present study should be considered. First, the novel variants in LPL, APOA5 and GPIHBP1 identified in the present cohort were not functionally assessed. However, the combination of in silico prediction, absence from the control population, presence in the HTG phenotype and the presence of decreased LPL activity suggests the presence of a causal relationship. Second, we selected candidate genes for the present analysis which were known to affect LPL activity. However, in view of the focus on LPL, non-LPL-related causes may have been missed. It will be interesting to perform whole exome sequencing to elucidate novel genetic variants particularly in those patients in whom no LPL-related abnormalities have been identified. These studies are currently underway.

Clinical implications

With the exception of a low-fat diet, therapeutic strategies to lower TG levels in patients with severe HTG are characterized by a relative lack of efficacy. Conventional pharmacological therapies, other than omega-3 fatty acids, lower plasma TG levels predominantly by stimulation of LPL activity, thereby rendering these strategies less effective in the case of a severely dysfunctional LPL protein and/or its major cofactors. Due to the costs of genetic analysis and the low prevalence of severe HTG, in-depth diagnostic strategies are not routinely used in out-patient clinics. In the present study, we have shown that a molecular diagnosis is feasible and effective in carefully selected groups with severe HTG. By unravelling the underlying molecular ‘defect’, differential therapeutic strategies may be developed. As an example, in the case of a genetic defect in GPIHBP1, where LPL itself is unaffected, we successfully used heparin infusion to facilitate activating LPL in the absence of GPIHBP1. Consequently plasma TG levels were substantially reduced [13]. This illustrates the notion that stabilization of the LPL protein increases its activity in vivo, prevents inhibition of angiopoetin-like protein 4 (ANGPTL4) and can be used for the development of novel therapeutic strategies [34]. Of note, Gpihbp1^−/−, Angptl4^−/− double knockout mice have completely normal plasma TG levels [34]. In addition, LPL gene therapy has been used successfully in patients with genetic LPL deficiency; a follow-up study is currently ongoing to definitively confirm the long-term benefit and safety of this approach [30]. In the case of APOC2 or LMF1 deficiency, no clear treatment options, beyond strict dietary restrictions, are currently available. To prevent pancreatitis, plasmapheresis in patients with complete APOC2 deficiency has been included as an option in the latest guidelines [35]. Finally, patients with severe HTG might benefit from the development of novel strategies to target TG-related processes, such as mRNA inhibition of apo C-III, small-molecule inhibitors of acylCoA:monoacylglycerol acyltransferase (MGAT), acylCoA:diacylglycerol acyltransferase (DGAT) 1 and 2, and antibodies against ANGPTL4 and fibroblast growth factor (FGF)21.