Comparative analysis of mutations in the LDLR gene

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To facilitate the mutational analysis of the LDLR gene and promote the analysis of the relationship between genotype and phenotype, in 1997 we created a software package along with a computerised database: UMD-LDLR. For each mutation, information is provided at several levels: at the gene level (exon and codon number, wild type and mutant codon, mutational event, mutation name), at the mRNA level (size, processing), at the protein level (wild type and mutant amino acid, affected domain, activity, mutation class), and at the personal level (ethnic background, age, sex, body mass index and familial history of coronary heart disease). The software package contains routines for the analysis of the LDLR database that were developed with the 4th dimensionR (4D) package from ACI. The use of the 4D SGDB gives access to optimised multi-criteria research and sorting tools to select records from any field. Moreover, 13 routines were specifically developed (Varret et al. 1997, 1998, Villeger et al. 2002, Beroud et al. 2005, www.umd.be/LDLR/).

The aim of this study was to analyse these four mutation groups at the molecular, biological and clinical level.

4.1. Analysis of LDLR mutations at the molecular level

4.1.1. Frequency of mutational events

DNA substitutions are of two types: transitions are interchanges of two-ring purines (A>G and G>A) or of one-ring pyrimidines (C>T and T>C) and, therefore, involve bases of similar shape; transversions are interchanges of purine for pyrimidine bases, which involve exchange of one-ring and two-ring structures. Therefore, there are twice as many possible transversions as there are transitions. However, among human diseases-causing substitutions, transitions (63%) are observed more frequently than transversions (37%) (Cooper and Krawczak 1990).

Accordingly, in the LDLR gene, missense mutations due to transitions (55.9%, 459/821) are more frequent than substitutions due to transversions (42.5%, 349/821) (Figure 3). Like exonic mutational events, small DNA variations at the splice site are substitutions (92.4%, 122/132) or small deletions/insertions (9.1%, 12/132). Again, among the intronic substitutions, transitions (59.8%, 73/122) are observed more frequently than transversions (40.1%, 49/122) (Figure 3). Interestingly, in the LDLR gene, the ratio of transversion/transition is different for nonsense mutations. The transversions are the more frequent mutational event leading to a stop codon (52.7%, 77/146) compared to transitions (47.3%, 69/146) (Figure 3).

Transitions Transversions

Figure 3. Molecular events frequency of the different groups of mutations. Values are given in % of each event within each group of mutation.

Because of the constraints mediated by the genetic code, transition A>G and transversion A>C, G>C cannot be at the origin of a stop codon. Thus, only two transitional events (G>A

and C>T) and 6 transversional events (A>T, C>A, C>G, G>T, T>A and T>G) lead to a stop codon, which means that half of the transitional events and a quarter of the transversional events are not involved in nonsense mutations. These constraints can explain the observed difference in the ratio of transversion/transition between missense and nonsense mutations.

However, the ratio of transversion/transition is consistent with the one observed for human diseases-causing substitutions (Cooper and Krawczak 1990) when the three groups of mutations are taken together (missense, nonsense and splice). Altogether, transitions (55.9%, 601/1076) are observed more frequently than transversions (44.1%, 475/1076).

4.1.2. Distribution of the substitutions in the 18 exons of the LDLR gene

The expected number of mutations in each exon is estimated by the 'Stat exons' tool of the UMD software according to the size and the composition (mutability of each codon) of each exon (Béroud et al. 2000 and 2005). This analysis enables the detection of a statistically significant difference between observed and expected mutations.

For exons 1, 5, 8 and 10 to 14, all types of substitutions are distributed as expected. There is a significant excess of all substitutions (missense and nonsense) within exons 3 and 4 (Table 1), indicating discrete mutational hot-spots and underlining the essential role played by the encoded domains in protein function. Exon 3 encodes the second LR motif of the ligand binding domain in the LDL receptor. To date, there is no data revealing a more essential function of this LR motif when compared to the six others. Exon 4 encodes the three central LR motifs (LR3, LR4 and LR5) of the ligand binding domain in the LDL receptor. The LR5 motif have been shown to be the only one of the seven LR motifs to be able to bind the two ligands of the receptor, apo B and apo E, while the 6 other motifs only bind apo B (Russel et al. 1989). Thus, the mutations affecting this motif are associated with a more severe alteration of lipoprotein catabolism and, therefore, have a higher tendency to be selected by FH definition criteria. There is a significant deficit of all substitutions (missense and nonsense) within exons 15 and 16 (Table 1) indicating discrete mutational cold-spots. Exon 15 encodes the O-linked sugar domain of the LDL receptor that has been shown to have no significant functional activity (Davis et al. 1986). To date, there is no explanation as to the observed deficit of substitutions within exon 16 which encodes the membrane-anchoring domain that is essential to the attachment of the receptor to the cell membrane.

The two types of exonic substitutions (missense and nonsense) are differently distributed in exons 2, 6, 7, 9, 17 and 18 of the LDLR gene (Table 1). Missense mutations are the only ones presenting a significant excess in exons 6 and 9 and a significant deficit in exons 17 and 18 (Table 1), maybe reflecting a bias in this analysis due to the different number of mutations of each type. Nonsense mutations are less numerous than missense mutations, a significant difference is thus less probably obtained for nonsenses than for missenses. Nevertheless, these observations indicate discrete mutational hot-spots within exons 6 and 9 and discrete mutational cold-spots within exons 17 and 18. Exon 6 encodes the last LR motif of the ligand binding domain in the LDL receptor. To date, there is no data revealing a more essential function of this LR motif when compared with the six others. Exon 9 encodes the NH2-

terminal part of the EGF-like domain which is rich in YWTD repeats which are essential for the correct folding of the receptor at the cell surface. To date, there is no explanation as to the observed deficit of substitutions within exons 17 and 18 encoding the COOH-terminal part of the membrane-anchoring domain and the cytoplasmic tail, which are essential for the attachment of the receptor to the cell membrane and in the endocytosis of the protein.

In exon 2, we observed a significant deficit of missenses and a significant excess of nonsenses (Table 1). Exon 2 encodes the first LR motif of the ligand binding domain in the LDL receptor. To date, there is no data revealing a more or less essential function of this LR motif when compared with the six others.

Interestingly, nonsense mutations are the only ones that present a significant excess in exon 7 of the LDLR gene (Table 1). This excess relies upon the high frequency of the c.1048C>T, p.Arg350X mutation, formerly called FH-Fossum. Indeed, this mutation is reported in 9 apparently unrelated patients from different geographic origins: Norway (Solberg et al. 1994), the Netherlands (Lombardi et al. 1995), the U.K. (Day et al. 1997), Poland (Gorski et al. 1998), Germany (Thiart et al. 1998), Canada (Gaudet et al. 1999), Japan (Yu et al. 2002), Denmark (Damgaard et al. 2005) and Spain (Brusgaard et al. 2006). In the absence of haplotypes demonstrating a common ancestor, these mutational events are supposed to be recurrent and to correspond to a mutational hot-spot in the LDLR gene.

Exon

Expected mutations

Observed missenses

Observed nonsenses

Observed exonic substitutions

Expected mutations missenses

(%)

%

significance

%

significance

%

significance

1

2,6

1,7

ns

2,7

ns

1,9

ns

2

5,0

2,5

< 0.01

11,6

< 0.001

3,9

ns

3

4,8

6,4

< 0.05

6,8

< 0.05

6,5

< 0.02

4

14,9

20,5

< 0.001

20,5

< 0.001

20,5

< 0.001

5

4,8

4,4

ns

3,4

ns

4,3

ns

6

4,9

7,0

< 0.01

5,5

ns

6,8

< 0.01

7

4,7

5,2

ns

8,9

< 0.02

5,7

ns

8

5,0

5,3

ns

4,8

ns

5,2

ns

9

6,6

11,2

< 0.001

4,1

ns

10,1

< 0.001

10

8,7

7,3

ns

6,2

ns

7,1

ns

11

4,7

4,9

ns

4,8

ns

4,9

ns

12

5,2

6,4

ns

2,1

ns

5,7

ns

13

5,6

5,3

ns

2,1

ns

4,8

ns

14

5,9

6,4

ns

9,6

ns

6,9

ns

15

6,4

1,6

< 0.001

2,1

< 0.05

1,7

< 0.001

16

2,8

1,5

< 0.05

0,0

< 0.05

1,3

< 0.01

17

6,1

2,3

< 0.001

4,8

ns

2,7

< 0.001

18

1,3

0,1

< 0.01

0,0

ns

0,1

< 0.001

Table 1. Distribution of the different exonic substitutions throughout the 18 exons of the LDLR gene.

Table 1. Distribution of the different exonic substitutions throughout the 18 exons of the LDLR gene.

4.2. Analysis of LDLR mutations at the biological level

4.2.1. Functional classes of LDLR gene's mutations

Mutations in the LDLR gene have been classified into 5 functional groups based on the characteristics of the mutant protein produced and analysed in patients' fibroblasts (Hobbs et al 1992):

Class 1 mutations disrupt the synthesis of the LDL receptor and no precursor is produced (null alleles).

Class 2 mutations block transport to the Golgi apparatus: mutations are reported in class 2A when a complete defect in transport to the cell membrane is observed and in class 2B when receptors are transported at a detectable - but markedly reduced - rate.

Class 3 mutations produce proteins that reach the membrane but fail to bind the LDL.

Class 4 mutations produce a receptor that binds the lipoprotein but which cannot be internalised. The mutations affecting the cytoplasmic domain alone are classed 4A, while those also affecting the membrane-spanning region are classed 4B.

Class 5 mutations block the acid-dependant dissociation of the receptor and the ligand in the endosome, an essential event for receptor recycling.

The link between the functional class type of the mutation and the severity of the disease has been established, and patients carrying a class 1 mutation are more severely affected than those with a mutation from another functional group (Hobbs et al 1992). In the UMD-LDLR database, among the 288 single nucleotide mutations with available data concerning the functional group, 42.0% (121/288) are class 2B, 31.9% (92/288) are class 1, 13.5% (39/288) are class 5, 7.6% (22/288) are class 2A, 3.8% (11/288) are class 4A and 1.0% (3/288) are class 3. Class 1 mutations are mainly nonsense and frameshift mutations (66.3% nonsenses, 30.4% frameshifts and 3.3% missenses) and 62% of them are localised in exons 2 to 6, encoding the ligand binding domain for one half and in exons 7 to 14 encoding the EGF-like domain for the other half (Figure 4). Class 2B mutations are mainly missense mutations (92.6% missenses and 7.4% frameshifts) and 71% of them are localised in exons 2 to 6, encoding the ligand binding domain (Figure 4). Class 5 mutations are mainly missense mutations (95% missenses and 5% splice site mutations) and 95% of them are localised in exons 7 to 14, encoding the EGF-like domain (Figure 4). Class 2A, 3 and 4A mutations are mainly missense mutations (59% missenses, 22% nonsenses and 19% frameshifts) and 67% of them are localised in exons 7 to 14, encoding the EGF-like domain. As expected, the localisation of these different classes of mutations is consistent with the functional definition of each class. The higher prevalence of mutations at the origin of truncated proteins (nonsenses and frameshifts) within the class 1 functional group is consistent with the expected null allele effect of these kinds of mutations. Altogether, these observations are globally in agreement with the admitted dogma according to which mutations leading to a protein of abnormal size (nonsense, frameshift and splice) are at the origin of a more severe phenotype than missense mutations.

Figure 4. Distribution of the different mutations according to the three main functional classes.

4.2.2. LDL receptor activity

In the UMD-LDLR database, the LDL receptor activity measured in patients' fibroblasts is available for 91 single nucleotide mutations: assays were performed for 24 heterozygote carriers, 22 homozygote carriers and 45 compound heterozygotes.. For homozygote carriers of a missense mutation, the mean LDL receptor activity is 8.7% rather than 2.7% for carriers of a mutation leading to a protein of abnormal size (nonsense, frameshift and splice) (Figure 5). For heterozygote carriers of a missense mutation, the mean LDL receptor activity is 33.2% rather than 19.8% for carriers of an abnormal-protein mutation. Moreover, a gradient can be drawn for compound heterozygotes with a mean LDL receptor activity of 13.3%, 7.3% and 3.6% for carriers of two missense mutations, one missense and one abnormal-protein mutation and two abnormal-protein mutations respectively (Figure 5). Once again, these observations are globally in agreement with an admittedly more severe phenotype for mutations leading to a protein of abnormal size when compared with missense mutations. However, missense mutations in the LDLR gene are associated with a larger spectrum of LDL receptor activity in fibroblasts (from 2% to 67% for heterozygotes and from 2% to 22.5% for homozygotes) when compared with mutations leading to a protein of abnormal size (from 2% to 47% for heterozygotes and from 2% to 11% for homozygotes).

Figure 5. LDL receptor activity in fibroblast from mutation carriers. The values are expressed as % of LDL binding compared with the values obtained for normocholesterolemic subjects. M: missense. N: null allele (frameshift, splice, nonsense).

4.3. Analysis of LDLR mutations at the biochemical/clinical level

4.3.1. Plasmatic lipid levels among LDLR gene mutations carriers

Among the 1061 unique events included in the UMD-LDLR database, lipid values are available for only 307 of them (29%), corresponding with 25 homozygote carriers and 282 heterozygote carriers of different molecular events within the LDLR gene (Table 2). According to the biochemical definition of familial hypercholesterolemia, triglycerides and HDL-cholesterol levels were within the normal range while the total- and LDL-cholesterol levels were elevated. As expected for a co-dominant disease, the total- and LDL-cholesterol levels were higher for homozygote mutation carriers than for molecular heterozygotes. No differences were observed between the four groups of mutations (missenses, frameshifts, splice sites and nonsenses), suggesting a similar effect of missense and mutations leading to a protein of abnormal size (nonsense, frameshift and splice) on the biochemical expression of the disease. Furthermore, no differences were observed among the distribution of totaland LDL-cholesterol levels among the four groups of mutations (Figure 6).

Figure 6. Distribution of total- and LDL-cholesterol plasmatic levels for heterozygotes carriers of a missense (M), a frameshift (F), a splice site (S) or a nonsense (N) mutation in the LDLR gene.

_HDL-Cholesterol LDL-Cholesterol Total Cholesterol Triglycerides

Heterozygotes

_HDL-Cholesterol LDL-Cholesterol Total Cholesterol Triglycerides

Heterozygotes

Missense N

133

144

152

137

Mean (SD)

1.31 (0.51)

7.50 (2.38)

9.50 (2.18)

1.66 (0.94)

Frameshift N

60

63

73

64

Mean (SD)

1.21 (0.34)

7.84 (2.05)

9.89 (2.22)

1.39 (0.89)

Splice N

22

25

30

24

Mean (SD)

1.28 (0.41)

7.17 (2.08)

9.56 (2.20)

1.49 (0.54)

Nonsenses N

24

24

27

27

Mean (SD)

1.17 (0.40)

7.74 (1.64)

9.43 (1.53)

1.46 (0.73)

Homozygotes

Missense N

13

15

14

12

Mean (SD)

1.04 (0.41)

15.55 (4.96)

17.39 (4.49)

1.42 (0.72)

Frameshift N

3

3

3

2

Mean (SD)

0.66 (0.21)

16.01 (1.17)

17.43 (0.93)

1.23 (0.04)

Splice N

3

3

5

4

Mean (SD)

0.67 (0.16)

15.25 (1.79)

18.06 (4.74)

1.34 (0.17)

Nonsenses N

2

2

2

2

Mean (SD)

0.87 (0.52)

17.54 (0.37)

19.56 (0.76)

2.00 (1.27)

Table 2. Mean plasmatic lipid levels for heterozygotes and homozygote carriers of missense, frameshift, splice site or nonsense mutations in the LDLR gene. Values are in mmol/L.

Table 2. Mean plasmatic lipid levels for heterozygotes and homozygote carriers of missense, frameshift, splice site or nonsense mutations in the LDLR gene. Values are in mmol/L.

4.3.2. Clinical expression of familial hypercholesterolemia among LDLR gene mutation carriers

Of the 1061 unique events reported in the UMD-LDLR database, clinical data is available for only 230 of them (22%) including 25 homozygote carriers and 215 heterozygote carriers of different molecular events within the LDLR gene (Table 3). This clinical data concerns tendinous cholesterol deposits - such as xanthomas - and the diagnosis of premature coronary artery disease (CAD). Tendinous xanthomas are more frequently observed for the carriers of a mutation leading to a protein of abnormal size rather than for the heterozygotes for a missense mutation (Table 3). Once more, this observation is in agreement with the admitted dogma according to which mutations leading to a protein of abnormal size (nonsense, frameshift and splice) are at the origin of a more severe phenotype than are missense mutations. However, no differences were observed for the occurrence of CAD between missenses and those mutations leading to a protein of abnormal size (Table 3). This latter observation suggests a similar effect with regard to missense and mutation leading to a protein of abnormal size (nonsense, frameshift and splice) in the clinical expression of the disease.

Missenses_Frameshifts, Splice sites, Nonsenses

Sex ratio (M/F)

1.06 (83/78)

1.09 (60/55)

Age (mean years ± SD)

39.6 ± 17.5

36.8 ± 14.9

N

Yes (%) No (%)

N

Yes (%)

No (%)

CAD

100

58 42

99

52

48

Tendinous xanthomas

106

50 50

109

65

35

Table 3. Clinical expression of familial hypercholesterolemia for heterozygotes carriers of different mutations in the LDLR gene.

Table 3. Clinical expression of familial hypercholesterolemia for heterozygotes carriers of different mutations in the LDLR gene.

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