uva-dare (digital academic repository) familial

18
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) UvA-DARE (Digital Academic Repository) Familial hypercholesterolemia: the Dutch approach Huijgen, R. Link to publication Citation for published version (APA): Huijgen, R. (2012). Familial hypercholesterolemia: the Dutch approach General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 08 Feb 2018

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Page 1: UvA-DARE (Digital Academic Repository) Familial

UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

Familial hypercholesterolemia: the Dutch approach

Huijgen, R.

Link to publication

Citation for published version (APA):Huijgen, R. (2012). Familial hypercholesterolemia: the Dutch approach

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 08 Feb 2018

Page 2: UvA-DARE (Digital Academic Repository) Familial

Chapter 6

Cardiovascular Risk in Relation to

Functionality of Sequence Variants in

the Gene Coding for the Low-Density

Lipoprotein Receptor

A study among 29,365 individuals tested for 64 specific LDLR sequence variants

Roeland Huijgen, Iris Kindt, Joep C. Defesche and John J.P. Kastelein

European Heart Journal 2012;33(18):2325-2330.

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Chapter 6

106

AbSTRACT

Background: A plethora of mutations in the LDL-receptor gene (LDLR) underlie the clinical phenotype of familial hypercholesterolemia (FH). For the diagnosis of FH, it is important, however, to discriminate between pathogenic and non-pathogenic mutations. The aim of the current study was to assess whether true pathogenic mutations were indeed associated with the occurrence of coronary artery disease (CAD) when compared to non-functional variants. The latter variants should not exhibit such an association with CAD.

Methods: We assessed 29,365 individuals tested for the 64 most prevalent LDLR variants. First, we determined pathogenicity for each of these sequence variants. Subsequently, a Cox-proportional hazard model was used to compare event-free survival, defined as the period from birth until the first coronary artery disease (CAD) event, between carriers and non-carriers of LDLR mutations.

Results: Fifty-four sequence variants in the LDLR gene were labeled as pathogenic and 10 as non-pathogenic. The 9,912 carriers of a pathogenic LDLR mutation had a shorter event-free survival than the 18,393 relatives who did not carry that mutation; hazard ratio 3.64 (95%CI: 3.24 to 4.08; p<0.001). In contrast, the 355 carriers of a non-pathogenic LDLR variant had similar event-free survival as the 705 non-carrying relatives; hazard ratio 1.00 (95%CI: 0.52 to 1.94; p=0.999).

Conclusions: These findings with respect to clinical outcomes substantiate our criteria for functionality of LDLR sequence variants. They also confirm the CAD risk associated with FH and underline that these criteria can be used to decide whether a specific sequence variant should be used in cascade screening

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Cardiovascular risk and functionality criteria for the LDLR

107

6

InTRODuCTIOn

Familial hypercholesterolemia (FH) is a frequent autosomal co-dominant disorder of lipoprotein metabolism, with 1 in 500 persons affected with heterozygous FH in most Western countries.1 Patients with FH have elevated plasma low-density lipoprotein-cholesterol (LDL-C) levels and an increased risk of premature coronary artery disease (CAD).1, 2 Defects in genes that code for proteins involved in hepatic clearance of LDL-C underlie this hereditary disorder.1 In fact, more than a 1000 different mutations in the genes coding for the LDL-receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are now known to cause FH.3

The identification of a mutation that underlies FH in a particular family enables genetic testing of family members for the presence of the same mutation and makes it possible to initiate effective medical management before the cardiovascular consequences of FH become clinically manifest. This notion has led to the implementation of a nationwide genetic cascade screening program for FH in the Netherlands and approximately 27,000 subjects with FH have been found and treated since 1994.4

However, as widely appreciated, not every sequence variant in LDLR, APOB or PCSK9 results in an FH phenotype. If a novel sequence variant in one of those genes is identified in a patient with a clear clinical FH diagnosis, one has to rely on in silico or in vitro studies to ascertain pathogenicity. However, in vitro studies are cumbersome and therefore rarely performed. Also, the in silico results do not always correspond with the clinical observations.5 An alternative strategy is to perform cascade screening for such a novel variant and to identify new carriers of that specific variant to determine whether these carriers express an FH phenotype. Accordingly, we recently validated specific criteria that can discriminate pathogenic from non-pathogenic mutations, using lipoprotein and lipid levels as well as the use of lipid-lowering medication.5 Since then, collected a much larger cohort of carriers of non-pathogenic variants in order to assess possible consequences of these variants on occurrence of cardiovascular disease.

In the present study, we compared cardiovascular risk between individuals with established pathogenic FH mutations and those with sequence variants in LDLR that we consider non-pathogenic. Here, we present our results.

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Chapter 6

108

METHODS

In this observational study we used a stepwise approach. First, we applied our criteria to assess functionality of all variants we have identified in LDLR in order to expand the number of individuals with pathogenic and non-pathogenic variants. Second, we compared CAD risk between carriers and non-carriers of all non-pathogenic variants and, similarly, of all pathogenic mutations.

Study populationThe functionality criteria and event free survival were assessed in individuals who were screened by genetic testing in the period between January 1994 and December 2010. Of these subjects, lipoprotein profiles, an extensive history of cardiovascular disease, medication use, and specific carrier status were collected at the time of molecular diagnosis. We excluded index patients from the current analysis to avoid clinical sampling bias. The cascade screening program was launched by the Ministry of Health of the Dutch Government and approved by the National Ethics Committee. All participants gave written informed consent for genetic analysis.

Criteria to test functionalityThe three criteria to establish functionality of a specific DNA variant have been described before 5 and were: (1) a mean LDL-C level above the 75th percentile for age and gender in untreated individuals carrying such a mutation, (2) statistically significantly higher mean LDL-C levels in untreated carriers compared to untreated non-carriers, and (3) a statistically significant higher percentage of medication users among carriers compared to non-carriers at the time of molecular screening. We considered a mutation to be non-pathogenic when none of the three criteria were met.

All genetic variants had to be tested in at least 50 untreated subjects per variant. Additionally, the in silico analyses were applied to all prevalent variants by using the Alamut software (version 2, Interactive Biosoftware, Rouen, France: SIFT and PolyPhen prediction). Mutations were described according to the nomenclature as proposed by den Dunnen and Antonarakis,6 and we used the description based on the effect of the mutation on the protein.

Coronary artery diseaseCAD was defined as a history of one of the following non-fatal cardiac outcomes: a) sudden cardiac arrest; b) myocardial infarction; c) coronary artery bypass graft, or d)

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Cardiovascular risk and functionality criteria for the LDLR

109

6

percutaneous transluminal coronary angioplasty. CAD event-free survival was defined as the period from year of birth until the year of the first CAD event or censoring at the moment of genetic screening.

Statistical analysisDifferences in LDL-C levels between subgroups were compared by means of the unpaired Student t-test. Proportions of subjects using lipid-lowering medication were compared with the chi-square test. We compared survival of mutation carriers with that of family members without mutations using Kaplan-Meier survival analysis. A Cox-proportional hazard model was used to compare CAD risk between the carriers and non-carriers. The non-carriers served as reference group. We adjusted for gender, hypertension, diabetes, smoking and body mass index. The Cox-proportional hazard analyses were repeated in R, taking also family ties into account. Sensitivity and specificity were calculated for the variants for which in silico analysis could be performed, using the described criteria as a gold standard for pathogenicity. 5 A two sided p-value < 0.05 was considered statistically significant. Data were analyzed with SPSS for Windows 16.0.2 (Chicago, IL).

Results

Test of functionality of new sequence variantsIn our study cohort, we extended previous findings with 12 sequence variants that had been tested in more than 50 untreated subjects just by 2010.5 All these variants reside in the LDLR and are presented in Table 1.3, 7-11 Three variants involve exonic deletions, whereas the other mutations result in amino acid substitutions.

Table 2 summarizes the in silico analysis and the assessment according to the criteria for all 12 variants. As observed, 11 mutations are pathogenic, whereas G701S (exon 14) is not pathogenic.

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Chapter 6

110

Tab

le 1

: Nom

encl

atur

e of

mut

atio

ns in

LD

LR

Loc

atio

nn

ucl

eoti

de

chan

gen

ew n

omen

clat

ure

Old

nom

encl

atu

re b

Ref

eren

cec

Exo

n 1

c.1A

>G

p.M

et1V

alM

-21V

1, 3

Exo

n 3

c.26

8G>

Ap.

Asp

90A

snD

69N

2E

xon

4c.

622G

>A

p.G

lu20

8Lys

E18

7K1

Exo

n 4

c.66

2A>

Cp.

Asp

221G

lyD

200G

1E

xon

6c.

858C

>A

p.Se

r286

Arg

S265

R1

Exo

n 9

c.12

43G

>C

p.A

sp41

5His

D39

4H1

Exo

n 9

c.12

97G

>T

p.A

sp43

3Tyr

D41

2Y1

Exo

n 9

c.13

29G

>C

or

G>

Tp.

Trp4

43C

ysW

422C

1E

xons

12-

18c.

1705

+?_

2580

+?d

elD

elet

ion

exon

s 12

-18

16 k

b de

leti

on o

f exo

n 12

-18

(Cat

ania

-1)

2, 4

Exo

n 14

c.21

01G

>A

p.G

ly70

1Ser

G68

0S2

Exo

n 16

c.23

11+

?_23

90+

?del

Del

etio

n ex

on 1

62.

0 kb

del

etio

n ex

on 1

6 (P

adov

a-2)

2, 5

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ns 1

6-18

c.23

11+

?_25

80+

?del

Del

etio

n ex

ons

16-1

88.

0 kb

del

etio

n ex

on 1

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2

Num

beri

ng o

f the

nuc

leot

ides

of t

he L

DL

R g

ene

was

bas

ed o

n th

e cD

NA

, wit

h +

1 be

ing

the

A o

f the

AT

G t

rans

lati

on in

itia

tion

cod

on. a N

ew n

ame

repr

esen

t nu

mbe

ring

of

the

codo

ns w

ith

the

init

iati

on c

odon

is

1. b O

ld n

ame

repr

esen

t nu

mbe

ring

of

the

codo

ns w

ith

init

iati

on c

odon

is

-21

for

LD

LR

. Ref

eren

ce s

eque

nce

LD

LR

: Gen

Ban

k A

cces

sion

NM

_000

527.

3. c R

efer

ence

s of

the

mut

atio

ns i

dent

ified

in

the

Net

herl

ands

: (1)

Fou

chie

r et

al

., H

um G

enet

109

:602

–615

, 20

01;

(2)

Fouc

hier

et

al.,

Hum

Mut

at 2

6:55

0–55

6, 2

005;

(3)

Lom

bard

i et

al.,

Cli

n G

enet

51:

430

-431

, 19

97;

(4)

Ber

toli

ni e

t al

., A

TV

B 1

9:40

8-41

8, 1

999;

(5) B

erto

lini

et

al.,

AT

VB

15:

81-8

8, 1

995.

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Cardiovascular risk and functionality criteria for the LDLR

111

6

Tab

le 2

: Mut

atio

ns w

ith

findi

ngs

of in

sili

co r

esul

ts a

nd o

f the

app

lica

tion

of c

rite

ria

to a

sses

s pa

thog

enic

ity

In si

lico

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sIn

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o ob

serv

atio

nW

itho

ut c

hole

ster

ol lo

wer

ing

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icat

ion

All

All

FH+

FH-

FH+

FH-

Sequ

ence

var

iant

SIFT

Pol

yPhe

nN

pLD

LLD

L-C

aLD

L-C

apb

%M

ed%

Med

p

M1V

not

tol.

prob

ably

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± 1

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2 ±

1.2

<0.

001

576

<0.

001

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Nno

t to

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ly51

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8 ±

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3.0

± 0

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021

5317

0.01

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± 1

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2 ±

1.1

0.00

149

14<

0.00

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not

tol.

prob

ably

7289

4.9

± 1

.52.

9 ±

1.0

<0.

001

5221

0.00

1S2

86R

not

tol.

prob

ably

8484

4.5

± 1

.23.

0 ±

1.0

<0.

001

6313

<0.

001

D41

5Hno

t to

l.be

nign

5081

4.3

± 1

.33.

0 ±

0.8

<0.

001

3615

0.04

1D

433Y

not

tol.

prob

ably

5096

5.9

± 1

.43.

3 ±

0.9

<0.

001

6021

0.00

4W

443C

not

tol.

prob

ably

9095

5.4

± 1

.43.

0 ±

0.9

<0.

001

6212

<0.

001

Del

etio

n ex

ons

12-1

8-

-60

976.

4 ±

0.9

3.1

± 1

.0<

0.00

163

9<

0.00

1G

701S

not

tol.

poss

ibly

5036

2.9

± 0

.73.

1 ±

0.7

0.67

3117

0.17

Del

etio

n ex

on 1

6-

-80

965.

5 ±

0.8

3.0

± 1

.1<

0.00

185

9<

0.00

1

Del

etio

n ex

ons

16-1

8-

-87

925.

3 ±

1.6

2.9

± 1

.0<

0.00

167

23<

0.00

1

a LD

L-C

leve

ls (m

mol

/l) a

re e

xpre

ssed

as

mea

n ±

sta

ndar

d de

viat

ion.

b p-v

alue

s ba

sed

on c

ompa

riso

n of

LD

L-C

leve

ls b

etw

een

untr

eate

d ca

rrie

rs a

nd n

onca

rrie

rs.

The

in

sili

co a

naly

ses

coul

d on

ly b

e pe

rfor

med

for

mut

atio

ns t

hat

resu

lt i

n an

am

ino

acid

sub

stit

utio

n an

d no

t fo

r de

leti

ons.

Abb

revi

atio

ns f

or S

IFT:

no

t tol

., no

t tol

erat

ed. F

or P

olyP

hen:

pos

sibl

y, p

ossi

bly

dam

agin

g; p

roba

bly,

pro

babl

y da

mag

ing.

Oth

er a

bbre

viat

ions

: FH

+, L

DL

R m

utat

ion

carr

iers

; FH

-, n

onca

rrie

rs o

f an

LD

LR

var

iant

; LD

L-C

, lo

w-d

ensi

ty l

ipop

rote

in c

hole

ster

ol;

p, p

-val

ue;

pLD

L, m

ean

perc

enti

le f

or a

ge a

nd g

ende

r; %

Med

, pe

rcen

tage

cho

lest

erol

-low

erin

g m

edic

atio

n us

ers.

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Chapter 6

112

Risk of coronary artery disease based on functionalityAll tested mutations combined amounted to a total of 64 sequence variants in the LDLR gene that could be labeled as either pathogenic or non-pathogenic.5 The characteristics of these variants including the in silico findings are shown in supplemental table 1. The total cohort consisted of 29,365 individuals, who were tested for the presence of one particular LDLR mutation (Table 3). This constitutes 83.3% of the overall population tested in the cascade screening program. Of these 29,365, 1,060 (3.6%) individuals were tested for one of the 10 non-pathogenic sequence variants, of whom 355 subjects carried that variant.

As can be expected, carriers of pathogenic mutations were more often treated with cholesterol lowering medication compared to carriers of non-pathogenic variants in LDLR (4,193/9,912 (42%) vs. 38/355 (11%); p<0.001). Also, the mean levels (± SD) of the highest total cholesterol levels measured over lifetime were significantly higher in the carriers of a pathogenic mutation than in those identified with a non-pathogenic variants (9.5 ± 3.1 vs 6.2 ± 4.3 mmol/L; p<0.001).

Figure 1 and 2 provide Kaplan Meier curves for CAD event free survival for individuals tested for pathogenic and non-pathogenic LDLR variants. The 9,912 carriers of a pathogenic LDLR mutation had on average a shorter event free survival than the 18,393 relatives who did not carry that mutation. The hazard ratio associated with carriership was 3.64 (95% confidence interval: 3.24 to 4.08; p<0.001) (Figure 1). In contrast, the Kaplan Meier curves overlapped between carriers of the non-pathogenic sequence variants and the relatives that did not carry the variants (Figure 2). In particular, the risk of CAD was similar for both carriers and their unaffected relatives: the hazard ratio was exactly 1.00 (95% confidence interval: 0.52 to 1.94; p=0.999). The analyses described above were also performed with R statistics - taking family ties into account - and these analyses yielded results that were in essence similar (data not shown).

The in silico analyses could be performed for 34 pathogenic mutations and 9 non-pathogenic variants according to the criteria,5 as shown in supplemental table 1. Of these 43 variants, 33 were amino acid substitutions that were not tolerated according to SIFT, indicating a non-pathogenic nature. The associated specificity was 0.22 (2 out of 9) and the sensitivity was 0.97 (33 out of 34). Polyphen identified 13 variants as benign, i.e. non-pathogenic. Consequently the specificity was 0.44 (4 out of 9) and the sensitivity 0.74 (25 out of 34).

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Tab

le 3

: Cha

ract

eris

tics

of t

he s

tudy

pop

ulat

ion L

DL

R m

utat

ion

yes

noP

atho

geni

c by

cri

teri

aal

lye

sno

all

p#

Num

ber

10,2

679,

912

355

19,0

97A

ge y

ears

**37

(22-

51)

37 (2

2-51

)42

(23-

55)

45 (3

1-57

)<

0.00

1M

ale

sex

n (%

)4,

941

(48%

)4,

773

(48%

)16

8 (4

7%)

9,06

1 (4

7%)

0.27

Bod

y m

ass

inde

x kg

/m2 *

23.7

± 4

.823

.6 ±

4.8

24.4

± 4

.724

.5 ±

4.5

<0.

001

Hig

hest

tot

al c

hole

ster

ol e

ver

mm

ol/L

* 9.

5 ±

3.1

9.5

± 3

.06.

2 ±

4.3

5.9

± 3

.7<

0.00

1St

atin

use

at

gene

tic

scre

enin

g n

(%)

4,23

1 (4

1%)

4,19

3 (4

2%)

38 (1

1%)

1,77

4 (9

%)

<0.

001

Cor

onar

y ar

tery

dis

ease

n (%

)69

8 (7

%)

683

(7%

)15

(4%

)67

6 (4

%)

<0.

001

**m

edia

n (b

orde

rs o

f qua

rtil

es),

*mea

n ±

sta

ndar

d de

viat

ion,

# p-v

alue

for

com

pari

son

of a

ll L

DL

R m

utat

ion

carr

iers

ver

sus

all n

on c

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.

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Figure 1: Kaplan Meier curves for individuals tested for pathogenic LDLR mutations

Carrier of pathogenic LDLR mutation in red and non-carrier in blue

Figure 2: Kaplan Meier curves for individuals tested for non-pathogenic LDLR sequence variants

Carrier of non-pathogenic LDLR mutation in red and non-carrier in blue

creo
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Cardiovascular risk and functionality criteria for the LDLR

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6

DISCuSSIOn

In this study, we first applied a set of specific criteria to determine functionality of the 64 most prevalent LDLR sequence variants used in the screening program in our country.5 Almost 30,000 individuals were tested for one of these variants and, as expected, carriers of pathogenic FH mutations had consistently elevated LDL-C levels. Moreover, FH patients with a pathogenic mutation had a more than 3 fold higher hazard ratio for CAD compared to unaffected relatives.

In contrast, lipid levels and the risk of CAD in individuals carrying a non-pathogenic LDLR variant were similar to those of their healthy relatives. As such, this provides further evidence that the proposed criteria to determine non-pathogenicity did label sequence variants correctly.

Only patients with a strong clinical suspicion of genetic FH are referred for analysis of LDLR mutations in the Netherlands. Once a novel sequence variant is identified in such an index patient, the clinical characteristics of the index patient will evidently not reveal whether that variant is functional or not. In fact, we showed previously that the clinical FH diagnostic scores were in essence not different for the index patients in whom an established pathogenic mutation was discovered from the index patients with a novel non-pathogenic variant in the LDLR.5 Thus, when in vitro tests are lacking and no segregation data is available yet, one often has to rely on the in silico analyses to decide whether further cascade screening should be performed for the novel LDLR variant. Table 1 and supplemental table 1 show that the findings in silico not correspond well with the co-segregation data, with a specificity below 50%. For example, the p.Gly701Ser mutation (c.2101 G>A) was identified as non-pathogenic rather convincingly by co-segregation while the SIFT and Polyphen indicated the amino acid substitution to be not tolerated and possibly damaging, respectively. Conversely, clinical data on the p.Glu208Lys mutation (c.662 G>A) showed that this mutation is clearly pathogenic, while the in silico analyses suggested that this was a non-pathogenic sequence variant. Thus, the clinical data from the index patients referred for genetic analysis combined with the in silico results are often not conclusive for determining functionality for novel LDLR sequence variants.

Therefore, in case the newly discovered sequence variant in LDLR was identified in a patient with a strong clinical suspicion of FH and the in silico analyses also hinted at pathogenic nature, the further approach was to include it in the genetic cascade screening. After a robust number of individuals was been tested for a variant that was assumed to cause FH, that variant was re-evaluated based on the criteria we proposed.5

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We advocate not to discard a mutation from inclusion in a screening program based on a pedigrees of less than 50 persons.5 In addition, three recent publications show that certainly not all carriers of a confirmed pathogenic LDLR mutation show an FH phenotype.12-14

Previously, most other reports on non-pathogenic variants in LDLR focussed on single mutations. Based on in silico, in vitro or co-segregation analyses or a combination of these, authors then concluded on the functionality of that specific variant.15-17 The sample size of the pedigrees that harboured such variants was often modest. In contrast, in our pooled analyses, we were able to assess a large dataset of LDLR variants that all were initially thought to be pathogenic and were therefore used for genetic screening in relatives. In fact, at the current time more than 500 different variants in LDLR have been identified in the Netherlands.18 Of the 29,365 individuals tested for the 64 most prevalent LDLR sequence variants, 355 (1.2%) were identified to carry a variant that was initially thought to cause FH, but was later shown not to be pathogenic. Thus, re-evaluation of functionality of sequence variants seems a reasonable approach to select only those FH mutations for genetic cascade screening that are associated with increased LDL-C levels, and consequently increased CAD risk.

Not surprisingly, we found that sequence variants in LDLR that did not lead to hypercholesterolemia did not affect CAD risk. In fact, we recently measured carotid intima-media thickness (IMT) in subjects with pathogenic LDLR mutations who had untreated LDL-C levels below the 75th percentile. Their IMT values were similar to those in unaffected relatives and less than those in genetic FH patients with hypercholesterolemia.12

The strength of the present study is that we could directly compare CAD risk between carriers of an LDLR variant and unaffected relatives. Several factors other than non-pathogenicity could potentially explain the lack of phenotypic expression of a LDLR mutation. These include healthy life style or co-incidence of neutralizing mutations.19-21 However, non pathogenicity of the sequence variants is by far the most likely explanation, because risk of CAD was not increased in individuals that carried these variants.

A potential limitation of our event-free survival analysis is that statin use at the time of genetic diagnosis differs between the groups. Particularly, statin use was more prevalent in the group of FH patients with a pathogenic mutation, compared to unaffected relatives and carriers of a non-pathogenic variant. As a consequence, the natural course of FH has likely been altered in this group and the estimate of CAD risk will be an underestimation of the true risk. However, medication use was similar

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6

between individuals carrying a non-pathogenic variant and their unaffected relatives. As a consequence, the assessment of CAD risk in the group of interest, i.e. carriers of a non-pathogenic LDLR sequence variant, was not biased by medication use.

In conclusion, 10 out of 64 sequence variants in the LDLR gene are proven here to be non-pathogenic both in terms of lipids as well as with regard to event free CAD survival. We therefore have demonstrated that our clinical outcome findings have substantiated the criteria for functionality of LDLR sequence variants. They also confirm the CAD risk associated with FH and underline that these criteria can be used to decide whether a specific sequence variant should be used in cascade screening.

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Chapter 6

118

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REFEREnCES

1. Goldstein JL, Hobbs HH, Brown MS. In: The metabolic and molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 2001: 2863-2913.

2. Huijgen R, Vissers MN, Defesche JC, Lansberg PJ, Kastelein JJ, Hutten BA. Familial hypercholesterolemia: current treatment and advances in management. Expert Rev Cardiovasc Ther 2008;6(4):567-581.

3. Leigh SE, Foster AH, Whittall RA, Hubbart CS, Humphries SE. Update and Analysis of the University College London Low Density Lipoprotein Receptor Familial Hypercholesterolemia Database. Ann Hum Genet 2008;72:485-498.

4. Umans-Eckenhausen MA, Defesche JC, Sijbrands EJ, Scheerder RL, Kastelein JJ. Review of first 5 years of screening for familial hypercholesterolaemia in the Netherlands. Lancet 2001;357(9251):165-168.

5. Huijgen R, Kindt I, Fouchier SW et al. Functionality of sequence variants in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in individuals with inherited hypercholesterolemia. Hum Mutat 2010;31(6):752-760.

6. den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000;15(1):7-12.

7. Bertolini S, Cassanelli S, Garuti R et al. Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1999;19(2):408-418.

8. Fouchier SW, Defesche JC, Umans-Eckenhausen MW, Kastelein JP. The molecular basis of familial hypercholesterolemia in The Netherlands. Hum Genet 2001;109(6):602-615.

9. Fouchier SW, Kastelein JJ, Defesche JC. Update of the molecular basis of familial hypercholesterolemia in The Netherlands. Hum Mutat 2005;26(6):550-556.

10. Lombardi P, Defesche JC, Kamerling SW, Kastelein JJ, Havekes LM. A novel mutation M-21V in exon 1 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Clin Genet 1997;51(6):430-431.

11. Bertolini S, Garuti R, Lelli W et al. Four novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15(1):81-88.

12. Huijgen R, Vissers MN, Kindt I et al. Assessment of carotid atherosclerosis in normocholesterolemic individuals with proven mutations in the low-density lipoprotein receptor or apolipoprotein B genes. Circ Cardiovasc Genet 2011;4(4):413-417.

13. Garcia-Garcia AB, Ivorra C, Martinez-Hervas S et al. Reduced penetrance of autosomal dominant hypercholesterolemia in a high percentage of families: Importance of genetic testing in the entire family. Atherosclerosis 2011;218 (2):423-430.

14. Huijgen R, Hutten BA, Kindt I, Vissers MN, Kastelein JJ. Discriminative Ability of LDL-Cholesterol to Identify Patients with Familial Hypercholesterolemia: A Cross-Sectional Study in 26,406 Individuals Tested for Genetic FH. Circ Cardiovasc Genet 2012;5(3):354-359.

15. Lombardi P, Sijbrands EJ, Kamerling S, Leuven JA, Havekes LM. The T705I mutation of the low density lipoprotein receptor gene (FH Paris-9) does not cause familial hypercholesterolemia. Hum Genet 1997;99(1):106-107.

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16. Ekstrom U, Abrahamson M, Sveger T, Sun XM, Soutar AK, Nilsson-Ehle P. Expression of an LDL receptor allele with two different mutations (E256K and I402T). Mol Pathol 2000;53(1):31-36.

17. Gudnason V, Patel D, Sun XM, Humphries S, Soutar AK, Knight BL. Effect of the StuI polymorphism in the LDL receptor gene (Ala 370 to Thr) on lipid levels in healthy individuals. Clin Genet 1995;47(2):68-74.

18. Defesche JC, Kastelein JJ. www.jojogenetics.nl. Website DNA diagnostic for Familial Hypercholesterolemia, last accessed May 2012.

19. Pimstone SN, Sun XM, du Souich C, Frohlich JJ, Hayden MR, Soutar AK. Phenotypic variation in heterozygous familial hypercholesterolemia: a comparison of Chinese patients with the same or similar mutations in the LDL receptor gene in China or Canada. Arterioscler Thromb Vasc Biol 1998;18(2):309-315.

20. van der Graaf A, Fouchier SW, Vissers MN et al. Familial defective apolipoprotein B and familial hypobetalipoproteinemia in one family: two neutralizing mutations. Ann Intern Med 2008;148(9):712-714.

21. Huijgen R, Sjouke B, Vis K et al. Genetic variation in APOB, PCSK9, and ANGPTL3 in carriers of pathogenic autosomal dominant hypercholesterolemic mutations with unexpected low LDL-C Levels. Hum Mutat 2012;33(2):448-455.