Enseñanza del Emprendimiento

Four mutations identified in the LPL gene of type I or CHL subjects were reproduced by in vitro mutagenesis. These were A158T, S193R, N291S and D9N. The effect of the mutations on LPL activity was studied using a transient expression system in cultured mammalian cells. Additionally, LPL mass in the culture medium was obtained for the D9N mutation. A control plasmid was not co-transfected with the mutant LPL constructs to standardize the measured enzyme activities for possible differences in transfection efficiency between dishes. In these conditions, the current system is at best a semi-quantitative assay.

The functional significance of two of the novel mutations identified in type I subjects, A158T and S193R, was confirmed by the in vitro mutagenesis experiments. Both mutations resulted in very low levels of activity. The fact that no other variants were detected by SSCP analysis in the LPL gene of the individuals carrying the A158T and S193R substitutions supports the interpretation that these mutations cause the phenotype. However, these results must be interpreted with caution since no LPL mass data was available for these two transfection experiments. Therefore, it cannot be ruled out that the transfections failed or were very inefficient due to the low transfectability of the LPL158 and 193 constructs.

Assuming a transfection efficiency similar to that of the wild-type construct, the low activity observed with the LPL158 construct agrees well with the in vivo data. LPL specific activity measured in subject SAk, homozygous for A158T, represented 3-4% of normal levels (Table 3.1). Recent modelling data suggests that A158T may alter a segment of the hydrophobic substrate binding pocket (van Tilbeurgh et al., 1994). This might restrict access of the substrate to the active site, which would be expected in turn to decrease the turnover rate and would be measured as lower specific activity.

The situation is more complex for the S193R mutation. Subject LA is a compound heterozygote and carries a second defective allele with a 2kb insertion which

has been reported to abolish correct mRNA splicing and hence LPL synthesis (Langlois et al., 1989). Yet, adipose tissue LPL activity for this individual ranges from 8-17% of levels measured in age-matched subjects (Lithell et al., 1978). This differs by several fold from the values obtained in the two transfection experiments and suggests the latter are an underestimate, possibly due to lower transfection efficiency. Alternatively, it is possible that the effect of this mutation is modulated in a tissue-specific manner such that it is less severe in adipose tissue and is not accurately reflected in COS cells. Brunzell and colleagues (1980) have described patients with adipose tissue-specific LPL deficiency although the molecular basis for these observations remains unclear.

Lower LPL activity and mass were consistently observed in the medium from Asn9 transfected cells compared to cells transfected with the wild type construct, although the decrease in activity was slightly more marked (range 17-40% for activity and 6-32% for mass). There was some variation in the results from the in vitro

expression studies. This was not surprising, since for several reasons the transient expression system used was not ideal for testing relatively small differences; namely the absence of a true internal control for the transfection efficiency of the plasmids and the large amount of inactive LPL protein. Although the number of cells per plates used was kept constant, there was no control for transfection efficiency. However, this issue is at least partially addressed by the use of multiple dishes which are randomized to different constructs, by combining data from several plates and by the consistency of the results in repeat experiments.

Secreted LPL-Asn9 appears to have only slightly reduced function. The heparin- Sepharose chromatography profile also demonstrated that the ability to bind heparin is preserved and the affinity appears unchanged. This suggests that the lower mass and activity in the medium is not caused by impaired release from the cell surface. This is not surprising since it is proposed that the main heparin-binding domains are located

between amino acids 270-305 (Hata et al., 1993; van Tilbeurgh et al., 1994), which are predicted to be distant from the N-terminal sequence in the tertiary structure of LPL. The data from these two sets of experiments imply that the Asn9 substitution may impair post-translational processes or secretion from the cell. This might be expected to lead to some accumulation of LPL within cells, which could not be determined accurately in these experiments due to the low sensitivity of the current transfection assay. These issues could be resolved by cell fractionation and metabolic labelling experiments in permanently transfected cell lines.

Finally, the N291S variant is of great interest because it is the first LPL mutation with a major effect on LPL activity which appears to be common both in hyperlipidaemic subjects (type I by this study, type III HLP by Ma et al., 1993c) and in the general population (chapter 4). The lower activity observed with the LPL291 construct (range 31.7 to 50.7%) has confirmed the report of Ma and colleagues. We have shown that this decrease appears to be caused largely by the lability of the mutant enzyme. The loss of activity is probably caused by the rapid dissociation of the active LPL dimer (Peterson et al., 1992). In agreement with this, a high level of LPL monomers has recently been reported by Reymer et al. (1995) in the media from cells transfected with the Ser291 allele. In addition, the dénaturation assay provides an explanation for the apparent inability of heparin to effect the release of LPL activity from the surface of LPL291 cells in the first transfection experiment. This result was somewhat unexpected as the mutation did not involve a positively charged amino acid which could bind to a surface heparan-sulphate molecule. In fact, it is likely that the mutant enzyme is released rapidly (and normally) by heparin but is inactivated after 24 hrs in the culture medium. Overall, these results suggest that residue 291 may play a role in the maintenance of a stable LPL dimer conformation.

6 . HETEROZYGOUS EXPRESSION O F LPL GENE MUTATIONS - POPULATION STUDIES.

Complete LPL deficiency due to two defective alleles at the LPL gene locus is a rare event with an estimated frequency of 1 : 1 0 0 0 0 0 0 and is expressed phenotypically as type I HLP (Brunzell, 1989). Based on this estimate, approximately one individual in 500 is expected to carry a non-functional LPL allele. In contrast, the studies presented in chapter 4 have identified two common, mild mutations (2-5% carrier frequency) in samples from the general population and from hyperlipidaemic subjects. Both the Asn9 and Ser291 variants (section 5.3 and 5.4) cause a decrease in LPL activity when expressed in vitro although this is more marked for the N291S substitution. In this chapter, the impact of these variants in vivo will first be investigated by examining LPL activity and lipid levels in unrelated heterozygous carriers. As a second step, the segregation of several LPL variants will be studied in families with FCHL and type I HLP.

Statistical analysis was carried out by Dr. Jackie Cooper for the Camberley and St.Andrews samples and by Dr. Viviane Nicaud for the ECTIM study.

6.1 Im pact of the D9N substitution on Tg and LPL levels in subjects from the UK, Sweden and the N etherlands.

6.1.1 Triglyceride levels.

The possible impact of the D9N substitution on lipoprotein metabolism was investigated by comparing lipid/lipoprotein traits and LPL activity (PHLA) in the carriers of the Asn9 allele identified in section 4.5 and in non-carriers from the same groups. Lipid levels, LPL activity where available, and biometrical data for the 55 Asn9

carriers that were identified in those groups are shown in Tables 6.1a & b.

In the first instance, the data from the two groups of healthy men recruited from the Camberley (England) and St. Andrews (Scotland) general practices were examined for the effect of the Asn9 allele. The 27 individuals with at least one Asn9 allele had significantly higher plasma triglycerides at entry in the study (2.25 vs 1.82, p < 0.02, 24% increase) than non-carriers (Table 6.2). When the individual data for triglycerides were plotted (Fig. 6.1) and compared to the Asp9 group mean, a considerable scatter in the levels of triglycerides emerged. One of the two homozygous individuals for Asn9 had relatively high levels while the other had levels slightly below the Asp9 group mean. The values were nevertheless distributed relatively evenly using the logarithmic scale and the higher Tg seen in carriers was not simply due to a few outliers. There were no other significant differences in age, BMI, total chol, and apoB between the two groups.

For 22 of 27 Asn9 carriers and 631 non-carriers, complete lipid data were available for baseline and three subsequent annual measurements. In the group of non­ carriers, there was a small but significant decrease in plasma triglyceride levels over the three years (p = 0.001). A similar trend could be seen in the carriers but it did not reach statistical significance (p = 0.07). However, the higher plasma triglycerides seen at baseline in LPL-Asn9 carriers were maintained throughout the study period with the difference being significant at year 2 and overall (p = 0.005 and p = 0.01 respectively)(Fig.6 .2). The magnitude of the increase remged from 20 to 35% over the three years and averaged 27% overall. The slightly lower cholesterol levels seen in carriers at baseline (Table 6.2) was also maintained over time, but overall the difference was still not significant (5.35 _+ 0.18 mmol vs 5.66 +. 0.04, p = 0.1).

Table 6.1 Age, body mass index and lipid levels of individuals with the Asp9 to Asn substitution.

a) Patients

Sample Age BMI Chol Tg HDL-C LPL activity

(percentile)* Dutch combined 1 58 _ 8 . 0 6.95 0.82 _ hyperlipidémies 2 81 - 8.3 3.06 0.83 - 3 56 - 8.5 2 . 0 0 1 . 0 0 - 4 57 - 6 . 2 2.72 1.38 - F 5 40 - 7.9 2.74 0.78 - F 6 63 - 9.0 4.99 0.83 - 7 56 - _{6.7 7.06 0.63} - 8 44 - 10.3 4.70 0.90 - 9 44 - 16.1 21.60 0.58 - 1 0 52 - 4.9 2 . 0 2 1.08 - 1 1 - - 6.4 1.40 1.72 -

Swedish YMI study - 2053 37 2 0 . 2 0 8 . 2 1 1.52 1 . 6 6 38 (10)

patients F 2110 41 22.23 4.67 1.14 1.48 - 2119 42 25.73 6.98 1.72 1.14 48 (21) 2 1 2 0 38 28.65 6.79 1.83 1 . 0 0 - Swedish hyper- 71 - _{26.6 5.98 3.47 0.93 336 (74)} triglyceridaemics 1 0 2 - 2 2 . 8 7.36 4.81 1 . 2 1 169 (10) 1 2 0 - 35.3 5.84 6 . 1 0 0.79 217 (27) CX combined 13 56 27.5 7.8 3.45 0.82 60 (78) hyperlipidémies F 35 50 28.9 1 0 . 6 2.04 1.08 35 (26) 18 43 25.6 6 . 6 4.88 0.79 27 (8) 15 43 31.8 9.4 6 . 2 0 0.80 77 (84) 190

b) Control individuals

Sample Age BMI Chol Tg LPL Activity

(Percentile)' GP Practice- 142 54 25.1 5.90° 2.90° 2 2 0 SE England 312 43 30.1 5.90° 1.90° - 350 53 32.1 5.80° 0.82 - 416 58 17.2 6.80° 1.47 - 420 57 23.7 4.50 0.87 184 501 54 26.0 5.70 3.30° 127 527 54 22.3 5.20 2.03° - 589 55 25.3 4.80 2.18° - 643* 52 21.9 4.80 1.57 - GP Practice- 710 61 28.7 7.1° 1.59 Scotland 792 52 27.5 5.8° 3.28° - 836* 53 28.2 6.3° 4.45° - 906 52 23.0 5.3 1.04 - 1006 57 31.0 6.4° 2.79° - 1007 58 28.6 5.2 3.46° - 1 0 1 0 58 32.4 6 .2° 8.08° - 1017 59 26.3 3.5 1.31 - 1026 59 33.6 4.4 2.08° - 1029 59 26.5 6 .2° 2 .0 1° - 1044 59 29.1 4.1 3.96° - 1073 52 31.7 5.1 1.91° - 1076 60 29.3 4.8 ' 5.12° - 1129 52 27.5 6 .8° 5.19° - 1169 60 27.9 6 .6° 0.90 - 1329 57 25.2 5.8° 1.62 - 1345 60 25.5 5.0 3.42° - 1145 26.1 5.9° 2.15° Swedish 4094 52 26.4 7.59° 2.75° 65 (21) controls 4074 45 23.6 6.09° 1.65° 63 (17) Swedish NTG 1 1 2 - 2 2 . 0 4.91 1.2 2° 238 (30) Dutch 1 42 25.0 5.05 1 . 1 2 Controls 2 37 2 1 . 0 5.83° 1.51° - 3 48 2 0 . 0 5.79° 1.58°

* The difference in the range of values between the study groups is due to the use of different heparin doses (50 or 100 units/kg body weight) and different LPL activity assays. Activity is expressed as nmoles FFA/ml/min. F = females, * Individuals homozgous for the LPL-Asn9, ® at or above sample mean