New rare genetic variants of LMF1 gene identified in severe hypertriglyceridemia

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Abstract 48
Context: LMF1 (lipase maturation factor 1) gene encodes for a protein involved in lipoprotein 49 lipase and hepatic lipase maturation. Homozygous mutations in LMF1 leading to 50 hyperchylomicronemia are rare in the literature and a few additional rare LMF1 variants have 51 been described with poor functional studies.

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Objective: The aim of this study was to assess the frequency of LMF1 variants in a cohort of 53 385 hyperchylomicronemic patients without homozygous or compound heterozygous 54 deleterious mutations identified in LPL, APOA5, APOC2, GPIHBP1 genes, and to determine 55 their functionality.

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Methods: LMF1 coding variants were screened using dHPLC followed by direct sequencing. 57 In silico studies were performed with SIFT and PolyPhen-2 softwares, followed by in vitro 58 functional studies using HEK-293T cells co-transfected with vectors encoding human LPL 59 and LMF1 cDNA. LPL activity was measured in cell culture medium after heparin addition 60 using human VLDL-TG as substrate.   their functionality (7-9).

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The aim of the present study was to assess the frequency of LMF1 variants by         The optimal volume of medium sample (40 µl) was tested for sensitivity and linearity of the 196 LPL activity assay (supplemental figure 1 and 2). A strong correlation between the LPL 197 activity and the volume was found in the same way as the initial plasma assay (R=0.99, 198 p<0.01) (14) (supplemental figure 1). The LPL activity assay was linear between 2 and 6 199 hours after heparin addition (supplemental figure 2). The longer incubation time tested (6 200 hours) was conserved to have enough power to discriminate the effect of LMF1 variants.

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Inter-assay reproducibility was verified using an internal control; the variation coefficient was    (Table 1).

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The complete list of identified variants, including variants in adjacent intronic regions covered 234 by the genetic analyses, is available in Supplemental Table 3. The 10/18 missense variants, predicted to be "probably damaging" by PolyPhen-2 or 249 "deleterious" by SIFT by in silico studies, were selected for functional analyses. The 250 p.Thr143Met variant, predicted to be "possibly damaging", was added since it was associated 251 with p.Pro562Arg in a patient, in order to investigate the role of each variant (Table 1). The

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So far, only 3 nonsense LMF1 mutations were previously found in homozygous 316 patients and considered to be causally involved in hyperchylomicronemia (p.Tyr439Ter, 317 p.Tyr460Ter, p.Trp464Ter) (4-6). Several LMF1 missenses variants have been identified in 318 moderate or severe hypertriglyceridemic cohorts (7-9) but only one study provided functional 319 data for 7 missenses variants. None of these missenses variant had any significant impact on 320 in vitro LPL activity (7).

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In the present study, functional data are provided for 12 missense variants. Three of 322 them were already studied by Surendran et al. (p.Arg354Trp, p.Arg364Gln and p.Pro562Arg) 323 (7). Among the 10 new variants, 2 were found deleterious in vitro (p.Gly172Arg and 324 p.Arg537Trp). The two variants p.Arg354Trp and p.Arg364Gln, without deleterious effect in 325 Surendran's study (7), were shown to have a detrimental impact on the LPL activity in our 326 study. Moreover, we found a similar defect in LPL activity for both pTyr439Ter and  Consistently, the segregation study in the p.Trp464Ter proband family revealed that 372 her normotriglyceridemic sister and mother shared the same heterozygous mutation, in 373 addition to the p.Arg451Trp variant, found to be non-deleterious in our functional study. activity. It is noteworthy that, moderate HTG is a common feature in severe infectious state.

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Endotoxins and inflammatory cytokines such as TNF, IL-1, IL-2 or IL-6, have been involved 385 in increased VLDL hepatic production, decreased VLDL clearance and moreover altered LPL  The study has some limitations. For instance, due to their paucity it was not possible to 394 provide any association studies of these rare LMF1 variants with plasma TG in the general    Table 1 520