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Keywords:

  • Familial hypercholesterolemia;
  • LDLR;
  • locus specific variant database;
  • in silico pathogenicity prediction

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

Familial hypercholesterolemia (FH) is caused predominately by variants in the low-density lipoprotein receptor gene (LDLR). We report here an update of the UCL LDLR variant database to include variants reported in the literature and in-house between 2008 and 2010, transfer of the database to LOVDv.2.0 platform (https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR) and pathogenicity analysis. The database now contains over 1288 different variants reported in FH patients: 55% exonic substitutions, 22% exonic small rearrangements (<100 bp), 11% large rearrangements (>100 bp), 2% promoter variants, 10% intronic variants and 1 variant in the 3' untranslated sequence. The distribution and type of newly reported variants closely matches that of the 2008 database, and we have used these variants (n= 223) as a representative sample to assess the utility of standard open access software (PolyPhen, SIFT, refined SIFT, Neural Network Splice Site Prediction Tool, SplicePort and NetGene2) and additional analyses (Single Amino Acid Polymorphism database, analysis of conservation and structure and Mutation Taster) for pathogenicity prediction. In combination, these techniques have enabled us to assign with confidence pathogenic predictions to 8/8 in-frame small rearrangements and 8/9 missense substitutions with previously discordant results from PolyPhen and SIFT analysis. Overall, we conclude that 79% of the reported variants are likely to be disease causing.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

The autosomal dominant condition familial hypercholesterolemia (FH)(OMIM 143890) is caused predominantly by variants in the low-density lipoprotein receptor gene (LDLR)(OMIM 606945) (Goldstein & Brown, 1989). Pathogenic changes in LDLR result in impaired uptake or processing of low-density lipoprotein (LDL) particles, which leads to elevated serum cholesterol and LDL levels, promotes atherosclerosis and increases the risk of coronary heart disease (Marks et al., 2003). As novel LDLR variants continue to be reported in FH patients, the utility of a locus-specific database for LDLR variants remains. The University College London (UCL) LDLR variant database was first set-up as an open access resource in 1996; it underwent an update in 2001 (Heath et al., 2001) and in 2008 Human Genetic Variation Society (HGVS) nomenclature was implemented, in silico pathological predictions were included, where appropriate, and the database was transferred to Leiden Open Source Variation Database (LOVD) platform version 1.1 (Fokkema et al., 2005; Leigh et al., 2008). This resource is of great clinical and research utility as it provides publicly available information on LDLR variants and their potential pathogenicity.

Here we report the latest update of the LDLR variant database with the addition of variants reported in the literature between 2008 and 2010 and 64 variants identified “in-house.” The database has been transferred to LOVD version 2.0, which provides additional functionality and ease of data submission and retrieval (Fokkema et al., 2011) and the database is now housed at Leiden University Medical Center, which provides automatic backup and software updates. Variants were assessed for their potential pathogenicity using open access in silico tools: PolyPhen 1 & 2 (Ramensky et al., 2002; Adzhubei et al., 2010), Sorting Intolerant From Tolerant (SIFT) (Ng & Henikoff, 2003), refined SIFT (Leigh et al., 2008), with further assessment of the structural impact using the Single Amino Acid Polymorphism database (SAAPdb)(Hurst et al., 2009) and local sequence conservation, calculated from a domain architecture-based sequence alignment from CATH-Gene3D (Lees et al., 2010) and conservation scoring with ScoreCons (Valdar, 2002). The predicted effects of intronic and synonymous variants on splicing were assessed using Neural Network Splice Site Prediction Tool (NNSSP) (Reese et al., 1997), SplicePort (Dogan et al., 2007) and NetGene2 (Hebsgaard et al., 1996).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

LOVD Platform

Existing and new variant data were loaded into the LOVD v.2.0 platform housed at Leiden University Medical Center (Fokkema et al., 2011). The nomenclature of all entries was assessed automatically using Mutalyzer (Wildeman et al., 2008) to check that current HGVS recommendations were adhered to. Additional columns listing new in silico pathological predictions were included. The updated database may be accessed via: https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR. Entries from the 2008 database will be migrated to the new platform from July 2012 and the 2008 database will continue to be available at http://www.ucl.ac.uk/ldlr and also via a link on the homepage of the updated database.

Identification of LDLR Variants Reported in the Literature

LDLR gene variants reported in the literature between 2008 and 2010 were identified using the terms: “Familial hypercholesterolemia, LDLR, & Mutation” and “Familial Hypercholesterolemia & Gene” in individual PUBMED searches.

LDLR Variants Reported “In-House”

All patients fulfilled the “Simon Broome” criteria for definite or possible FH (Marks et al., 2003). Patient details and the techniques used to identify the variants are listed on the LDLR database. All variants reported in the Centre for Cardiovascular Genetics were identified using a high-resolution melting technique and confirmed by sequencing as described (Whittall et al., 2010) and by the Great Ormond Street Hospital group using a combination of methods as described (Taylor et al., 2010).

In Silico Prediction of Variant Pathogenicity

Nonsense substitutions, frame-shifting small rearrangements and large rearrangements were not subjected to in silico analysis, as they are accepted to be pathogenic. The predicted effects of missense variants on LDLR function were assessed using the following open access software: (1) PolyPhen (Ramensky et al., 2002) and PolyPhen 2 (Adzhubei et al., 2010). The PolyPhen programs give predictions of “Benign,”“Possibly Damaging,” and “Probably Damaging.” PolyPhen 2 has two separate training datasets “Hum Div” and “Hum Var,” and prediction outcomes are labelled in the same way as for PolyPhen. (2) SIFT (Ng & Henikoff, 2003) and Refined SIFT (Leigh et al., 2008). Both SIFT programs predict whether an amino acid substitution would be “Tolerated” or “Not Tolerated.”

Where appropriate, the effects of variants on splicing were assessed using NNSSP, SplicePort and NetGene2 (Hebsgaard et al., 1996; Reese et al., 1997; Dogan et al., 2007). All programs give predictive scores for splice acceptor and donor sequences for wild-type and variant sequences. Variant results are presented as a percentage of the wild-type activity and changes were only considered to be significant if identified by all three programs. The effect of variants reported in the 5' untranslated sequences of LDLR on transcription were assessed using transcription factor binding affinities, site directed mutagenesis and standard luciferase assays (A. Khamis; unpublished data).

Additional In Silico Analysis of Variants

Additional in silico analyses were performed on in-frame small rearrangements and also on missense variants where predictions were discordant using the programs outlined above.

SAAPdb (Hurst et al., 2009)

The data pipeline analyses protein structure and constructs queries to link mutations with analysis rules. These rules include: functional residues, changes from glycine and proline, clashes (buried small-to-large), voids (buried large-to-small), hydrogen bonding, conserved residues, interface residues and other factors.

Analysis of conservation and structure

A set of peptide sequences with the same type and order of structural domains and approximately the same length (± 10%) were extracted from the Gene3D database (Lees et al., 2010) and aligned with the sequence for LDLR using the multiple sequence alignment programme, MAFFT (Katoh & Toh, 2008), with the –maxiterate 1000 and –globalpair options for high accuracy. Sequences that were missing a significant portion or contained many indeterminate residues were removed. Also, Gene Ontology assignments and UniProt descriptions for the homologues were extracted from Gene3D, and those not annotated as LDLRs were also removed (Cuff et al., 2009) (Table S1). Sequence conservation was calculated using ScoreCons (Lees et al., 2010) for the entire alignment, covering the whole length of LDLR. ScoreCons results are given on a scale with total conservation as one and no conservation as zero. The alignment was visualised using Jalview (Waterhouse et al., 2009). A second alignment of the sequence of PDB structure 1n7d against LDLR was used to map the conservation values to the Protein Data Bank (PDB) structure and visualised using PyMol version 1.3 for residues 65–714. Mutation Taster (Schwarz et al., 2010) and Project Hope (Venselaar et al., 2010) were also used where appropriate. However, Project Hope only provided additional information for the variant p.(Leu339Pro), and was therefore not included in the results for other variants.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

Novel LDLR Gene Variants

Following a systematic review of the literature and the inclusion of 64 variants identified “in-house,” a total of 223 novel variants were added to the UCL LDLR variant database. Of these variants, 117 are exonic substitutions (11 synonymous; 5%)(Table 1), 14 nonsense (6%)(Table S2), and 92 missense (41%)(Table S3 and Table 2). Four variants are in the 5' untranslated and proximal promoter region (2%)(Table S4), 38 variants are in intronic sequences (17%)(Table S5), 43 are small exonic rearrangements (<100 bp)(34 frame-shift; 16%) (Table S6) and eight in-frame (4%)(Table 3), 20 are large rearrangements (>100 bp) (9%), with defined breakpoints reported for eight of these (Table S7). And one variant is in the 3' untranslated sequence (Table S8).

Table 1.  In silico analysis of synonymous variants.
ExonAlleleProteinSplicePortNNSSPNetGene2 serverRemarksOverall in silico prediction
  1. Note: Effect of synonymous variants on normal splicing. For ease of identification the protein descriptions in this table do not follow HGVS recommendations of p.(=). Splicing scores represented as a percentage of wild type. A, splice acceptor site; D, splice donor site.

  2. *In vitro analysis supports pathogenic prediction for these variants (Defesche et al., 2008; Bourbon et al., 2007). Original references for variants can be found at: https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR.

3c.300C>Tp.(Asp100Asp)100 (exon 3A), 84 (exon 3D)100 (exon 3 A & D)100 (exon 3A & D)Minimal effect on splicingNonpathogenic
4c.621C>Gp.(Gly207Gly)100 (exon 4A & D)100 (exon 4 A & D)100 (exon 4A & D)No effect on splicingNonpathogenic*
4c.690C>Tp.(Asn230Asn)100 (exon 4A), 92 (exon 4D)100 (exon 4A), 105 (exon 4D)100 (exon 4A), 90 (exon 4D)Minimal effect on splicingNonpathogenic
8c.1185G>Cp.(Val395Val)100 (exon 8A), 127 (exon 8D)100 (exon 8A), 116 (exon 8D)100 (exon 8A & D)Minimal effect on splicingNonpathogenic
9c.1194C>Tp.(Ile398Ile)89 (exon 9A), 100 (exon 9D)60 (exon 9A), 100 (exon 9D)98 (exon 9A), 100 (exon 9D)Minimal effect on splicingNonpathogenic
9c.1216C>Ap.(Arg406Arg)102 (exon 9 A), 109 (novel A), 100 (exon 9D)100 (exon 9 A), 708 (novel A), 100 (exon 9D)136 (exon 9A), 86 (novel A), 100 (exon 9D)Minimal effect on splicingPossibly pathogenic*
10c.1503G>Ap.(Ala501Ala)100 (exon 9A & D)100 (exon 9A & D)100 (exon 9A & D)No effect on splicingNonpathogenic
11c.1635G>Tp.(Gly545Gly)113 (exon 11 A), 85 (exon 11 D)100 (exon 11 A & D)100 (exon 11 A & D)Minimal effect on splicingNonpathogenic
12c.1836C>Tp.(Ala612Ala)100 (exon 12A), 33 (exon 12D)100 (exon 12A & D)100 (exon 12A & D)Reduced affinity for exon 12 donor (Splice Port)Nonpathogenic
12c.1845G>Ap.(Glu615Glu)100 (exon 12A), 0 (exon 12D)100 (exon 12A), 0 (exon 12D)100 (exon 12A), 0 (exon 12D)Exon 12 donor site destroyedPathogenic
13c.1875C>Tp.(Asn625Asn)93 (exon 13A), 100 (exon 13D)100 (exon 13A & D)92 (exon 13A), 100 (exon 13D)Minimal effect on splicingNonpathogenic
Table 2.  In silico analysis of discordant missense substitutions.
ExonAlleleProteinPolyphenPolyphen 2 (Hum Div)Polyphen 2 (Hum Var)SIFTSIFTScore ConsSAAPMutation TasterSplice portNNSSPNetGene2Overall in silico prediction
  1. Note: Additional in silico analysis of discordant variants with three or four “nonpathogenic” predictions (from a combination of PolyPhen and SIFT programs). All prediction scores and values are as outlined in the text. A, splice acceptor site; D, splice donor site. Original references for variants can be found at: https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR.

4c.367T>Cp.(Ser123Pro)BenignBenignBenignNot toleratedNot tolerated0.616Disrupts H-bond, causes clash & introduction of prolineDisease causing (aa sequence change, protein feature)98 (exon 4A) 100 (exon 4D)100 (exon 4A) 100 (exon 4D)100 (exon 4A) 100 (exon 4D)Probably pathogenic
4c.599T>Gp.(Phe200Cys)BenignBenignBenignToleratedNot tolerated0.378Highly conserved sitePolymorphism (splice site changes, amino acid sequence changed)100 (exon 4A) 100 (exon 4D)100 (exon 4A) 100 (exon 4D)100 (exon 4A) 100 (exon 4D)Probably pathogenic
4c.632A>Tp.(His211Leu)Probably damagingBenignBenignNot toleratedTolerated0.847Hydrophobic side chain introduced on protein surfaceDisease causing (splice site changes, amino acid sequence change and protein features)100 (exon 4A) 98 (exon 4D)100 (exon 4A) 100 (exon 4D)100 (exon 4A) 100 (exon 4D)Possibly pathogenic
6c.940G>Ap.(Gly314Arg)BenignBenignBenignToleratedTolerated0.656No problems identifiedDisease causing (SNP, protein features affected, splice site changes)100 (exon 6A) 71 (exon 6D)100 (exon 6A) 52 (exon 6D)100 (exon 6A) 97 (exon 6D)Probably pathogenic
7c.1016T>Cp.(Leu339Pro)BenignProbably damagingBenignToleratedNot tolerated0.403Highly conserved site, causes clash & introduction of prolineDisease causing (protein features affected, splice site changes)117 (exon 7A) 119 (exon 7D)100 (exon 7A) 100 (exon 7D)100 (exon 7A) 100 (exon 7D)Probably pathogenic
12c.1834G>Tp.(Ala612Ser)BenignPossibly damagingProbably damagingToleratedTolerated0.710Introduction of hydrophilic residue in core of proteinPolymorphism (protein features affected, splice site changes)100 (exon12A) 34 (exon 12 D)100 (exon 12A) 100 (exon 12D)100 (exon 12A) 100 (exon 12D)Possibly pathogenic
15c.2296A>Gp.(Thr766Ala)BenignBenignBenignNot toleratedNot tolerated0.319Not applicableDisease causing (splice site changes, amino acid sequence change)100 (exon 15A) 93 (exon 15D)100 (exon 15A) 100 (exon 15D)100 (exon 15A) 100 (exon 15D)Probably not pathogenic
16c.2389G>Ap.(Val797Met)BenignBenignBenignToleratedNot tolerated0.545Not applicableDisease causing (protein features affected, splice site changes)82 (exon 16A) 0 (exon 16D)100 (exon 16A) 89 (exon 16D)100 (exon 16A) 89 (exon 16D)Probably pathogenic
17c.2399T>Ap.(Val800Asp)possibly damagingBenignBenignNot toleratedTolerated0.432Not applicablePolymorphism (aa sequence changed, protein features (might be) affected, splice site changes)108 (exon 17A) 100 (exon 17D)104 (exon 17A) 100 (exon 17D)155 (exon 17A) 100 (exon 17D)Probably pathogenic
Table 3.  In silico analysis of small in-frame rearrangements.
ExonAlleleProteinScoreConsPredicted effect on protein structureMutation TasterSplice predictionsOverall in silico prediction
  1. Note: All predictions and values for the in silico tools used are as outlined in the text. Splice predictions are only given where there has been a change from the wild-type score. Original references for variants can be found at: https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR.

4c.656_661delGCCCCGp.(Gly219_Pro220del)0.685, 0.416Disrupts calcium bindingPolymorphism (protein features lost)61% exon 4 donor (SplicePort)Probably pathogenic
4c.663_683dupp.(Asp221_Asp227dup)1.000, 1.000, 0.823, 0.985, 0.730, 0.931, 1.000Disrupts calcium bindingN/A109% exon 4 donor (SplicePort), 102% exon 4 donor (NetGene2)Probably pathogenic
4c.670_675delinsTTTp.(Asp224_Lys225delinsPhe)0.985, 0.730Disrupts calcium bindingDisease causing (protein feature affected, spice site changes)58% exon 4 donor (SplicePort), 97% exon 4 donor (NetGene2)Probably pathogenic
4c.681_683delCGAp.(Asp227del)1.000Disrupts calcium bindingDisease causing (several protein features affected & splice site changes)65% exon 4 donor (SplicePort), 99% exon 4 donor (NetGene2)Probably pathogenic
11c.1684_1686dupTGGp.(Trp562dup)0.918Disrupts calcium bindingPolymorphism (protein features lost)96% exon 11 donor (SplicePort)Probably pathogenic
12c.1776_1778delp.(Gly593del)0.611Disrupt tight packing of propeller blade 5Disease causing (aa sequence changed, protein features (might be) affected, splice site changes)143% exon 12 acceptor, 225% exon 12 donor (SplicePort)Probably pathogenic
13c.1870_1872delATCp.(Ile624del)0.472Disrupt tight packing of propeller blade 6Disease causing (protein feature affected, spice site)110% exon 13 acceptor (SplicePort) 92% exon 13 acceptor (NetGene2)Probably pathogenic
17c.2395_2397delp.(Leu799del)0.523Shortening of transmembrane domainDisease causing (protein feature alterations, splice site changes and listed as a SNP)125%, 110%, 260% exon 17 acceptor (NNSSP, SplicePort and NetGene2)Probably pathogenic

In comparison with the distribution of variants in 2008 (Leigh et al., 2008), greater percentages of variants were found in exons 10, 12, 13, 16, in the promoter and in intronic sequences in this study compared with 2008, nevertheless, novel variants were reported in every exon except 18 (Fig. 1) and all introns except intron 15. Chi-square tests revealed that there was no significant difference in the number of variants found according to domain (grouped exons 1–6, 7–14, 15–18) between the previously and newly collected data (P= 0.418), or the number of variants found in exons 3 and 4 in comparison with the remaining exons (P= 0.797).

image

Figure 1. (A) Percentage of LDLR variants reported previously (black bars) and in this study (excluding large rearrangements)(open bars) in all LDLR exons, including the promoter and intronic regions. Previously reported data is shown in black and newly reported data in white. More variants are reported in this study in the promoter, intron and exons 10, 12, 13 and 16 than previously reported. There is no significant difference between variants reported in exons 3 and 4 compared with the others (P= 0.797) or when grouped as exons 1–6, 7–14 and 15–18 (P= 0.418) or exons 1–4, 5–9, 10–14 and 14–18 (P= 0.088). (B) Using the alignment of functionally similar proteins with the same domain types, number and order conservation scores were calculated for each position with ScoreCons. Shown are the scores for the positions that align with LDLR, with the columns coloured according to the functional domains (see key). The very low conservation of the signal peptide region indicates that the alignment is sufficiently diverse to identify key conserved residues, the O-linked sugar region also exhibits very low conservation, the structural domains are all highly conserved.

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Pathogenicity Predictions of Variants

Exonic substitutions

All 14 nonsense variants reported here are considered to be pathogenic (Supporting Information Table S2). Synonymous variants are generally viewed as “silent,” however, depending on their position in the gene they may disrupt normal splicing, codon usage, mRNA folding and stability, which may adversely affect normal peptide synthesis (Sauna & Kimchi-Sarfaty, 2011). Analysis of the 11 synonymous variants reported here suggest that 9 would have no effect on splicing, as the affinity scores for the variant and wild-type sequences were not markedly different (Table 1). However, the variant c.1845G>A destroys the exon 12 donor splice site and is therefore likely to be pathogenic. Although the results for c.1216C>A at the native exon 9 acceptor site were only minimally changed, all three predictive programs identified a novel acceptor site with affinity levels equivalent to the wild-type. If this site were used it would result in a frame-shift and a truncated peptide (p.(His407Thrfs*7)) and could therefore be considered to be pathogenic.

Using PolyPhen 1 & 2 (version 2.1.0), SIFT, and Refined SIFT analyses, 73 missense substitutions were predicted to be pathogenic and 10 nonpathogenic (Table S3), although nine variants gave discordant results, with three or four “nonpathogenic” predictions (from a combination of PolyPhen and SIFT programs). These nine variants were analysed further (Table 2). Conservation scores were calculated for each position in the LDLR peptide with ScoreCons (Valdar, 2002), using the alignment of functionally similar proteins with the same domain types, number and order (Fig. 1B). These scores were used for analysing the degree of conservation of residues involved in missense variants and small rearrangements, and were applied to the three-dimensional (3D) model of the extracellular portion of LDLR to allow visualization of the conservation patterns in the peptide. In addition to the ScoreCons analysis, the nine missense variants with discordant results were also analysed with SAAPdb and Mutation Taster.

p.(Ser123Pro): SAAPdb analysis of p.(Ser123Pro) suggests that removal of the serine would destroy hydrogen bonds, and that introduction of a proline residue, which is larger and more rigid than serine, would further disrupt the LDL class 3A ligand-binding domain where this residue is located. These results are in agreement with those from Mutation Taster and lead us to suggest that this variant is probably pathogenic (Table 2).

p.(Phe200Cys): Located in the LDL-receptor class A5 repeat, p.(Phe200Cys) was designated as nonpathogenic by Mutation Taster. However, conservation analysis shows that this variant occurs in an environment of highly conserved residues with disulphide bonds around it (Fig. 2Ai). Therefore, introduction of an additional cysteine residue at this position could potentially result in the formation of disulphide bonds with cysteine residues in the region (positions 197, 204, or 209) (Fig. 2Ai). Formation of novel disulphide bonds would disrupt the wild-type configuration of this domain and would probably be pathogenic.

image

Figure 2. 3D diagrammatic representation of conservation score (residues 22–720) coloured to represent conservation scores (red: high, purple: moderate, blue: poor and black: no conservation). The residue of interest in each model is shown in green and does not reflect the level of conservation, the orientation chosen best shows the residue of interest. (A) Phenylalanine200 is poorly conserved (inset i) in a highly conserved environment, local disulphide bridges shown as green lines. (B) Histidine211 is highly conserved. (C) Glycine314 is on the surface of the protein, moderately conserved (inset i) and close to calcium cation (inset ii). (D) Leucine339 is on the surface of the protein and is poorly conserved (inset i and ii). (E) Ramachandran plot for p.(Leu339Pro), dark green areas show the favoured regions for prolines, whereas pale green shows acceptable areas. Triangles represent prolines present in the native structure (black in favoured regions, orange in acceptable regions, red in disfavoured regions). There are many prolines in disfavoured regions due to a poor quality structure. Leucine339 which is mutated to proline is shown as a blue square in a disfavoured region for proline. Unlike any of the disfavoured prolines in the native structure, it has a more negative phi angle. (F) Alanine612 is moderately conserved, it is buried within the structure and so cannot be visualised in the diagrammatic representation of conservation score.

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p.(His211Leu): Histidine211, which is also located in LDL-receptor class A5 repeat, (Fig. 2B) is highly conserved (ScoreCons 0.847), it is positively charged and polar whereas leucine is neutral and nonpolar. The difference in polarity and charge may impact on ligand binding and therefore this variant could possibly be pathogenic (Table 2).

p.(Gly314Arg): Glycine314 in the epidermal growth factor (EGF)-like 1 domain is not highly conserved (ScoreCons 0.656) (Fig. 2Ci) and analysis of the SAAPdb provides no evidence for the potential pathogenicity of p.(Gly314Arg). The 3D structure in the region revealed that glycine314 is next to a threonine residue that is involved in binding a bound calcium cation (Figs 2Ci and ii), it is possible therefore, that the substitution of the glycine with a positively charged arginine could affect the binding affinity of the calcium cation, although arginine is found at this position in other species (lizard and Chinese hamster). Examination of this variant at the DNA level reveals that the guanine at position 940 is the last residue in exon 6, and changing this to a thymine is predicted to reduce the affinity of the splice donor site to 71, 52, and 97% of wild-type (SplicePort, NNSSP and NetGene2, respectively) (Table 2). There are no other splice donor sites in the region with equal or greater binding affinities, and it is possible that normal spicing of exons 6 and 7 may be disrupted, therefore this variant could be pathogenic either as a result of the structural change or by disrupting splicing.

p.(Leu339Pro): The leucine339 in the EGF-like 1 domain, is located on the surface of the peptide and is poorly conserved across species (ScoreCons 0.403)(Figs. 2Di and ii), however, SAAPdb revealed that substitution of leucine with proline causes a small clash, and a Ramachandran plot (Fig. 2E) indicates that introduction of proline would be unfavourable for folding. Project Hope analysis of p.(Leu339Pro) suggested that Leu339 is also involved in several multimer contacts, and introduction of the smaller proline at this position may prevent these contacts being made. Overall, we predict that this variant is probably pathogenic.

p.(Ala612Ser): ScoreCons analysis (ScoreCons 0.710)(Fig. 1B) indicates that the β-propeller domain, which includes Ala612, shows a generally high degree of conservation (Fig. 2F); an average of 0.72 compared to the overall average of 0.64. Of note, the core region shows a very high degree of conservation, and many of the most conserved regions are buried, whereas the surface contains fewer highly conserved regions. Conservation analysis of the 3D structure shows that Ala612 is buried within the LDLR structure and lies within a highly conserved core structural element of the β-propeller domain, (residues 611–614 have the following ScoreCons scores: 0.708, 0.708, 0.780, 0.951, respectively). It is highly unlikely that changes will be easily tolerated in this region and may well affect the folding rate of the domain. Analysis of c.1834G>T, underlying p.(Ala612Ser), with SplicePort suggests that it reduces the affinity score at the exon 12 splice donor site to 34% of normal, (Table 2). This site already has a low affinity and a further reduction may interfere with normal splicing. However, it should be noted that NNSSP and NetGene2 did not identify this reduction. Overall, this variant is most likely to be pathogenic because of its influence on the structure of the peptide.

p.(Thr766Ala): Substitution of threonine766 in the O-linked glycosylation region will remove a sugar residue from the mature peptide. Furthermore, this substitution replaces a highly hydrophobic residue with the less hydrophobic alanine (hhHydrophobicity 0.52 and 0.11, respectively). However, this variant may not be pathogenic, as deletion of this region has been shown to have no effect on receptor function (Davis et al., 1986), although it is thought to maintain the LDL-binding domains at an appropriate distance from the cell surface (Hussain et al., 1999).

p.(Val797Met): Replacement of the strongly hydrophobic (hhHydrophobicity =–0.31) valine797 in the core of the transmembrane domain with the nominally hydrophobic (hhHydrophobicity =–0.1) methionine is likely to affect insertion of the peptide into the membrane and helix formation, thereby inhibiting anchoring of LDLR in the cell membrane. The substitution (c.2389G>A), responsible for p.(Val797Met) affects the last residue of exon 16; analysis of this variant with the splicing programs revealed that the affinity scores were reduced at the exon 16 donor site (0% SplicePort, 89% NNSSP and NetGene2)(Table 2). This is in agreement with experimental findings from the reporting group (Bourbon et al., 2009). Therefore, this variant is probably pathogenic because of its affect on splicing. Similarly, although the substitution c.2389G>T (p.(Val797Leu) was predicted by the PolyPhen and SIFT programs to be nonpathogenic (Table S3), it is also predicted to disrupt normal splicing (0% SplicePort, 82% NNSSP, 63% NetGene2) and therefore to be pathogenic.

p.(Val800Asp): The third transmembrane domain variant p.(Val800Asp), is also predicted to be pathogenic as the valine800 (hhHydrophobicity =–0.31) is replaced with the hydrophilic aspartic acid thereby potentially disrupting LDLR insertion into the membrane.

Promoter variants

Four variants were reported in the 5' untranslated promoter region, their impact on LDLR function will be reported elsewhere (A. Khamis; unpublished data). Based on this in vitro work, three were designated as pathogenic and one as benign (Supporting Information Table S4).

Intronic variants

Sixteen of the 38 intronic variants reported in this study are likely to be nonpathogenic, as in silico analysis reveals that their affinity scores either differed only slightly from wild-type (n= 14) or were the same as wild-type (n= 2) (Table S5). Conversely, 18 intronic variants are predicted to be pathogenic, as they resulted in complete destruction of the wild-type splice site (Table S5). Cryptic splice sites could be activated by two of these variants. c.1358 + 1G>T destroys the exon 9 splice donor site; were the cryptic splice site to be used it would result in a frame-shift p.(Ser453fs*2). Similarly, a cryptic splice acceptor site at c.1907 may be activated by variant c.1846–2A>C which would result in destruction of the wild-type exon 13 splice acceptor site, use of this cryptic splice site would also cause a frame-shift p.(Glu615fs*30). The potential use of cryptic splice sites in these variants would have pathogenic effects.

Four variants gave weaker evidence for pathogenicity with the splice prediction programs. The affinity scores for variant c.313 + 6T>C were reduced at the exon 3 donor site (Table S5). Bourbon et al. (2009), who reported this variant, showed experimentally that it resulted in the skipping of exon 3 and so it is probably pathogenic. In silico analysis of the variant c.941–13T>A, showed reduced affinity scores at the exon 7 splice acceptor site and the creation of a novel splice acceptor (Table S5); if this site were used, exon 7 would start at c.941–11 resulting in a reading frame-shift and termination of translation after 60 codons (p.(Gly314fs*60)). Similarly, c.2547 + 3G>C results in reduced affinity scores and there is a cryptic site nearby, which if used would also result in a reading frame-shift (p.(Ser849fs*6)(Table S5), however this variant has only been reported in the presence of c.798T>A p.D266E which is itself pathogenic (Chmara et al., 2010) and so c.2547 + 3G>C may not be pathogenic.

The affinity scores at the exon 10 acceptor site for the variant c.1359–5C>G were reduced to 74% (NNSSP and SplicePort) and 99% (NetGene2) of normal, which is inconclusive (Table S5). However, the reporting group (Bourbon et al., 2009) demonstrated in vitro that intron 9 is retained in the transcript from this variant; it is therefore predicted to be pathogenic.

Although the affinity scores at the exon 14 donor site remained unchanged in the variant c.2140 + 86C>G, a novel site was created, which according to Kulseth et al. (2010) is used and inserts 27 amino acids into the LDLR peptide and would therefore be pathogenic.

Small DNA rearrangements

Thirty-seven of the small rearrangements are considered to be pathogenic as they result in a reading frame-shift (Table S6). The eight in-frame small rearrangements reported in this study were subjected to in silico analysis was used in an attempt to predict whether they affect LDLR function (Table 3).

p.(Gly219_Pro220del), p.(Asp221_Asp227dup), p.(Asp224_Lys225delinsPhe) and p.(Asp227del): These variants affect residues within one of two hairpin loops in the class 5A repeat of the LDLR ligand-binding domain, which is stabilised by three disulphide bonds (cysteine residues 197 and 209, 214 and 231, 204 and 222). This domain includes the highly conserved D-x-S-D-E motif, spanning positions 224–228 (Kurniawan et al., 2000; Jeon et al., 2001; Jeon & Blacklow, 2005). Furthermore, residues 221 and 227 are among the four most highly conserved acidic residues directly involved in calcium coordination in LDLR (Kurniawan et al., 2000; Jeon et al., 2001; Jeon & Blacklow, 2005). Although the glycine and proline residues in p.(Gly219_Pro220del) are moderately and poorly conserved, respectively (ScoreCons 0.685, 0.416)(Fig. 3Ai), their deletion would alter the structure, disrupt H-bonding and prevent formation of the hairpin loop which requires a glycine (Fig. 3Aii). This would be likely to result in disruption of the local structure and opening up of the ends of the beta sheet strands linked by the loop. These residues also lie approximately 6Å from a calcium binding site and are likely to be required for its correct formation (Figs 3Ai and ii). Variants p.(Asp221_Asp227dup) and p.(Asp227del) will both destroy the D-x-S-D-E motif and are likely to distort the hairpin loop (Figs 3B and D). In the variant p.(Asp224_Lys225delinsPhe) not only are the highly and moderately conserved acidic residues aspartic acid and lysine deleted (Fig. 3Ci), they are replaced with the neutral and hydrophobic phenylalanine. As with the other variants in this region, their replacement is likely to disrupt calcium binding, which will in turn affect protein folding, break the hairpin loop and thereby alter the conformation (Fig. 3Dii). We suggest that these four variants are probably pathogenic.

image

Figure 3. 3D diagrammatic representation of conservation score (residues 22–720) coloured to represent conservation scores (red: high, purple: moderate, blue: poor and black: no conservation). The residue of interest in each model is shown in green and does not reflect the level of conservation, the orientation chosen best shows the residue of interest. (A) Glycine219 and proline220 are located on the surface of the LDLR protein. Glycine219 is moderately conserved and proline220 poorly conserved (inset i), both residues surround a calcium cation and form part of a hairpin (inset ii). (B) Residues from aspartate221 to aspartate227 are located on the surface of the protein, are mostly highly conserved (inset i, within the oval) and surround a calcium cation (inset ii). (C) Aspartic acid 224 is highly and lysine225 moderately conserved (inset i), both are located on the surface of the protein and surround a calcium cation (inset ii). (D) Aspartic acid 227 is located on the surface of the protein, is highly conserved and binds the calcium cation (inset i).

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p.(Trp562dup): Tryptophan562, buried within the peptide (Fig. 4A), is highly conserved and close to a calcium cation (Figs 4Ai–iii). Duplication of this residue in the variant p.(Trp562dup) is likely to disrupt coordination of the calcium cation (Figs 4Ai–iii) and is therefore probably or possibly pathogenic.

image

Figure 4. 3D diagrammatic representation of conservation score (residues 22–720) coloured to represent conservation scores (red: high, purple: moderate, blue: poor and black: no conservation). The residue of interest in each model is shown in green and does not reflect the level of conservation, the orientation chosen best shows the residue of interest. (A) Trytophan562 is located deep in the protein but is visible. It is highly conserved (inset i) and is located in the EGF domain (inset ii). (B) Glycine593 is located on the surface of the protein and is moderately conserved (inset i), it is at the end of a strand (inset ii). (C) Isoleucine624 is located on the surface of the protein, is poorly conserved (inset i) and forms part of a hairpin loop (inset ii).

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p.(Gly593del) and p.(Ile624del): Glycine593 and isoleucine624 are both in the β-propeller domain and form part of a hairpin loop in the fifth and sixth propeller blades, respectively (Figs 4B and C). These blades pack tightly against the C-terminal EGF module (Jeon & Blacklow, 2005), and deletion of these residues could disrupt the packing of these propeller blades, ultimately affecting displacement of the ligand from the ligand-binding region. Overall, we suggest that both of these variants are probably pathogenic.

p.(Leu799del): Leucine799 lies within the transmembrane domain of LDLR and deletion of this residue would shorten the transmembrane domain and may alter the structural conformation. This variant may therefore affect insertion of the peptide into the membrane, and is therefore probably pathogenic.

Large DNA rearrangements

Of the 20 large rearrangements reported here (Table S7), two were predicted to produce no peptides as the initiation codon and upstream sequences were deleted. Three of the variants were predicted to result in in-frame duplication of exons, five in in-frame deletion of exons and nine were predicted to produce truncated proteins. The remaining variant (c.2311 + 1941_*1216dup) was not predicted to affect the peptide length as it duplicates 725bp in the 3' untranslated sequence, however, this additional material may affect mRNA processing. It is therefore probable that all of the large DNA rearrangements are pathogenic.

3' untranslated variant

Analysis of c.2583 + 43G>A with Mutation Taster predicts that this variant is nonpathogenic.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

The results that we present in this paper show that although LDLR variants were first reported in FH patients in 1982 (Goldstein et al., 1982), novel LDLR variants continue to be reported in this group of patients by researchers from around the world. With over 1300 independent LDLR variants listed in the LDLR variant database so far, this is comparable with the number of variants reported in the genes responsible for other commonly occurring diseases such as cystic fibrosis (1902)(http://www.genet.sickkids.on.ca/Home.html), and haemophilia (1209) (http://www.hadb.org.uk/WebPages/PublicFiles/MutationSummary.htm).

The distribution of variants along the LDLR gene has altered slightly over the years, which is probably due to advances in high-throughput techniques that allow rapid and cost-effective whole gene screening. Previously, interest focussed on exons 3 and 4, which encompass the ligand-binding domain of LDLR, and the majority of variants were reported in these exons. However, there is now no significant difference (P= 0.80) between the proportion of variants found in exons 3 and 4 compared with the other exons, in previously (Leigh et al., 2008) and newly reported data. We also compared the proportion of variants reported per functional domain (the functional regions, as opposed to the structural domains discussed elsewhere in the paper), and found that there was no significant difference between variants found in each domain between previously and newly reported data (P= 0.418). Thus, variants are spread along the length of the LDLR gene and are not clustered in “disease-associated” domain(s).

To assess the clinical significance of the variants reported here, we have subjected them to standard and additional in silico analyses where appropriate. Pathogenic predictions (i.e., FH-causing) have been ascribed to all nonsense substitutions, frame-shifting small rearrangements and large rearrangements. Following analysis with PolyPhen and SIFT programs, 73 of the missense substitutions were considered to be pathogenic as they resulted in three or more adverse predictions, 10 were considered to be nonpathogenic, with five nondamaging predictions. The remaining nine missense substitutions, together with eight in-frame small rearrangements (which cannot be analysed with PolyPhen and SIFT programs) were all subjected to additional in silico analysis.

In the conservation and structure analysis we generated conservation scores for each residue in the LDLR peptide, represented graphically in Figure 1B. As may be expected, the highest levels of conservation are in the ligand-binding domains and the lowest in the signal peptide and the O-linked oligosaccharide region. Using the results generated by ScoreCons and Pymol, we have been able to construct a conservation model of the extracellular regions of the LDLR peptide, which has allowed us to visualise the residues of interest in the context of their environment in the mature peptide (Figs 2–4). Together with the information provided from the other analyses (SAAPdb, Mutation Taster, Project Hope, NNSSP, SplicePort and NetGene2), we have now assigned pathogenicity predictions with greater confidence to the remaining missense and small in-frame rearrangement variants (Tables 2 and 3). Of the nine previously ambiguous missense variants, six are now considered likely to be pathogenic, two possibly pathogenic and one is unlikely to be pathogenic (Table 2, Fig. 2). We consider that all of the small in-frame rearrangements are likely to be pathogenic using these methods (Table 3, Figs 3 and 4). It should be noted that 3D modelling could not be performed for the variants in the transmembrane domain, as the crystal structure for this region has not been resolved. However, alterations in this domain are likely to be pathogenic as they are predicted to adversely affect insertion of the peptide into the cellular membrane.

Analysis of the 11 synonymous substitutions reported in this study has revealed that although nine are unlikely to affect splicing, the remaining two: c.1216C>A and c.1845G>A are likely to disrupt normal splicing (Table 1). In the future, in silico analysis may be able to assess how synonymous variants influence transcription, translation and posttranslational processing by their effect not only on splicing, but also on codon usage, mRNA stability and translational speed (Sauna & Kimchi-Sarfaty, 2011). This is especially important in view of in vitro analyses which provide evidence for c.621C>G (p.(Gly207Gly)) and c.1216C>A (p.(Arg406Arg)) disrupting normal splicing (Bourbon et al., 2007; Defesche et al,. 2008) despite in the case of c.621C>G having no perceptible impact according to in silico predictions. In light of these observations, we encourage researchers to report and analyse these generally underreported “silent” variants.

Eighteen intronic variants reported in this study are predicted to be pathogenic as they destroy the wild-type splice sites (Table S5). Of the remaining 20 intronic variants, 16 are considered to be nonpathogenic as they result in little or no change in the NNSSP, SplicePort and NetGene2 affinity scores. The results for four variants were less conclusive (c.313 + 6T>C, c.941–13T>A, c.2547 + 3G>C, c.1359–5C>G), however, for c.313 + 6T>C and c.1359–5C>G the reporting groups have demonstrated experimentally that exon skipping and intron inclusion occurred in these variants, respectively (Bourbon et al., 2009; Chmara et al., 2010). These observations emphasise the value of wet lab analysis to assess the pathogenicity of such variants. Similar analysis is required to give conclusive results for the remaining variants. Furthermore, information regarding additional analyses that have been performed by reporting groups; such as segregation or in vitro studies may be easily accessed via a link to the original reference for each variant, these should always be followed up when considering the impact of variants in a clinical setting.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

In this update of the UCL LDLR variant database, we have added 223 novel LDLR variants. Pathogenic predictions have been ascribed to 183 (82%) of these variants. We have extended the usual range of in silico analyses to include detailed examination of potential structural changes. Although the combination of the standard and additional prediction methods outlined adds strength and validity to our predictions, it must be remembered that they are predictions and must be used with caution; experimental and/or patient family studies will be required to confirm or disprove these predictions. This is demonstrated in a report by Huijgen et al. (2012), who conclude that although in silico analysis predicts the variant p.Gly701Ser to be pathogenic, segregation data suggests that it is not. Our experience leads us to recommend that a range of in silico analyses be performed to reveal unexpected consequences of the variants on peptide structure and mRNA processing.

The UCL LDLR database continues to be a vital resource for clinicians and researchers alike, providing easy access to variant information, including pathogenic predictions. We would like to encourage researchers in the field to submit their variants to the database and we value being notified of any errors.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

SEH holds a Chair funded by the British Heart Foundation. SEH, SL and RW were supported by the BHF (PG08/008). CY is funded by NIH for the CSGID structural genomics initiative and CAO is funded by HEFSE.

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosure of Conflicts of Interest
  10. References
  11. Supporting Information

Table S1 List of proteins used to create the alignment for ScoreCons analysis.

Table S2 Nonsense substitutions.

Table S3 Missense substitutions with in silico analysis.

Table S4 Promoter substitutions.

Table S5 Intronic variants with in silico analysis.

Table S6 Small frame-shift rearrangements.

Table S7 Large rearrangements.

Table S8 3&apos; untranslated variant.

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AHG_724_sm_SupMat.doc409KSupporting info item

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