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The spectrum of premature atherosclerosis: from single gene to complex genetic
disorder
Trip, M.D.
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Trip, M. D. (2002). The spectrum of premature atherosclerosis: from single gene to complex genetic disorder
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Download date: 17 Jun 2017
Part I
Chapter 2
Molecular genetic testing in Familial
Hypercholesterolemia:
spectrum o f LDL receptor gene mutations
in the Netherlands
M Paola Lombardi1,4,
EgbertJW Redeker3Joep C Defesche', Sylvia WA Kamerling4,
2
Mieke D Trip , Marcel MAM Mannens3, Louis M Havekes4,JohnJP Kastelein1
Departments o f Vascular Medicine, 2 Cardiology and 3 Clinical Genetics, Academic Medical Centre,
Amsterdam, The Netherlands
4
TNO-Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands
Clin Genet 2 0 0 0 : 5 7 : 1 1 6 - 1 24
Part / Chapter 2
Abstract
M u t a t i o n s in the LDL receptor are responsible for familial hypercholesterolemia
( F H ) . At present, more than 600 m u t a t i o n s o f the LDL receptor gene are k n o w n t o
underlie FH. However, the array o f m u t a t i o n s varies considerably in different
p o p u l a t i o n s . Therefore, the delineation
o f essentially all LDL receptor gene
m u t a t i o n s in a p o p u l a t i o n represents a prerequisite for the implementation o f
n a t i o n - w i d e genetic testing for FH. In this study, the frequency and geographical
d i s t r i b u t i o n o f 13 k n o w n mutations were evaluated in a c o h o r t o f 1 223 FH patients.
W e identified 358 m u t a t i o n carriers, representing 29% o f the FH c o h o r t . Four
m u t a t i o n s ( N 5 4 3 H - 2 3 9 3 d e l 9 , 1 3 5 9 - 1 G - > , 31 3 + 1 G -> A and W 2 3 X ) occurred
w i t h a relatively high frequency, a c c o u n t i n g for 22.4% o f the entire study c o h o r t .
T w o o f these c o m m o n FH mutations ( N 5 4 3 H - 2 3 9 3 d e l 9 and 1359 - 1 G -> A)
showed a preferential geographic d i s t r i b u t i o n . Second, to further expand the array
o f LDL receptor gene mutations, we conducted m u t a t i o n analysis by denaturing
gradient gel electrophoresis (DGGE) in 141 children w i t h definite FH. A m u t a t i o n
was identified in 111 patients, involving 1 6 new single base substitutions and four
small deletions and insertions, which brings the number o f different FH-causing
m u t a t i o n s in our c o u n t r y up to 6 1 . O u r data indicate t h a t an estimate o f the
prevalence o f specific mutations, as well as the c o m p i l a t i o n o f a database o f all FHcausing m u t a t i o n s
in a given c o u n t r y , can facilitate selection o f the
most
a p p r o p r i a t e molecular diagnostic a p p r o a c h .
Introduction
Familial hypercholesterolemia (FH) is a c o m m o n a u t o s o m a l , d o m i n a n t l y inherited
disorder o f l i p o p r o t e i n metabolism 1 , caused, in the vast majority o f cases, by
m u t a t i o n s in the l o w density lipoprotein (LDL) receptor gene. Also, m u t a t i o n s in
the a p o l i p o p r o t e i n B100 gene are k n o w n t o cause a phenotype undistinguishable
from FH.2
Recently, evidence for the existence o f a t h i r d gene t h a t might be involved in an ' F H like' phenotype became available. 3
Historically, FH is diagnosed on typical clinical traits:elevation o f LDL cholesterol
up to twice the n o r m a l level, presence o f x a n t h o m a t a and a family history o f
premature coronary artery disease ( C A D ) . However, a clinical diagnosis o f FH is n o t
always unequivocal, especially in young patients in which physical stigmata are
30
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
often not present. In these cases, a molecular assay w o u l d be desirable for certain
diagnosis in families and for genetic counselling.
Therefore, the elucidation o f essentially all LDL receptor gene m u t a t i o n s in our FH
patients is a prerequisite for the development o f nation-wide genetic testing for FH.
At present, more t h a n 600 mutations o f the LDL receptor gene are known t o
underlie
FH w o r l d w i d e 4 " 6
However, the spectrum
of mutations
in
different
populations varies to a large extent. 7
Previously, a number o f LDL receptor mutations in the D u t c h FH p o p u l a t i o n were
reported, including ten large rearrangements 8 , 9 and 26 point m u t a t i o n s . 1 0 " 1 7 While
some m u t a t i o n s were also identified in other European countries 1 1 ' 1 2 ' 1 7 , the
majority were so far unique t o the Dutch FH p o p u l a t i o n .
In the present study, we first sought to identify prevalent LDL receptor gene
m u t a t i o n s and we therefore evaluated the frequency and geographical distribution
o f 13 k n o w n m u t a t i o n s in a c o h o r t o f 1 223 FH patients, dispersed t h r o u g h o u t the
low countries. Second, in order t o extend the spectrum o f LDL receptor gene
m u t a t i o n s , we conducted D N A analysis by denaturing gradient gel electrophoresis
(DGGE) in 141 heterozygous FH patients w i t h a definite clinical diagnosis based o n
internationally accepted criteria. 1
Material and methods
Patients
All patients were o f Dutch descent and met the criteria for a diagnosis
of
heterozygous FH: LDL cholesterol above the 95th percentile for sex and age,
presence o f t e n d o n x a n t h o m a t a and history o f premature atherosclerosis in the
patient or in a first-degree relative.
For m u t a t i o n analysis, 141 patients, referred to the Amsterdam Lipid Research
Clinic o f the Academic Medical Centre and the Slotervaart University Teaching
H o s p i t a l , were recruited. In all patients, the presence o f large rearrangements in the
LDL receptor gene 8 , 9 and o f t h e R3500Q m u t a t i o n o f t h e A p o B g e n e 2 was excluded.
For the assessment o f t h e frequency o f specific m u t a t i o n s , 1223 samples were
collected from 46 lipid clinics evenly distributed over the different regions o f t h e
country. All patients were referred to the regional lipid clinic by cardiologists,
internists and general practitioners (GPs), based on suspected lipid disorders. A
diagnosis o f FH was given according to uniform diagnostic criteria established
within the lipid clinic network. All patients were informed o f t h e o u t c o m e o f t h e test
31
Part 1 Chapter 2
by their GP. T o the best o f our knowledge, all patients, b o t h in the small group for
m u t a t i o n analysis as well as in the large c o h o r t for frequency o f known m u t a t i o n s ,
were n o t related.
D N A analysis
Genomic D N A was isolated from whole b l o o d samples, as previously described 1 8 .
For DGGE analysis, all 18 exons and the p r o m o t e r region o f the LDL receptor gene
were individually a m p l i f i e d from genomic D N A by PCR using Super Taq D N A
polymerase
(HT
Biotechnology,
Cambridge,
United
Kingdom)
as described
previously 1 1 , w i t h the following modifications. Exon 4 was amplified in t w o
overlapping fragments w i t h pairs o f primers as described by H o b b s et a l . 4 PCR
a m p l i f i c a t i o n o f the p r o m o t e r region was performed as described by Nissen et a l . 1 9
All fragments, except for the 3'-half o f exon 4, were amplified w i t h the same cycling
p r o t o c o l , w h i c h consisted o f 94°C 5 min and 32 cycles o f 94 °C 1 m i n , 5 5 ° C 30 s,
7 2 ° C 1.5 min in an O m n i G e n e Thermal cycler ( H y b a l d , A s h f o r d , United Kingdom).
T h e 3 ' - h a l f o f exon 4 required an annealing temperature o f 6 2 " C for PCR
a m p l i f i c a t i o n . DGGE analysis was conducted as previously described. 1 1 Exons
displaying aberrant m i g r a t i o n patterns on DGGE gels were amplified and directsolid phase D N A sequencing was performed as described by H u l t m a n et a l . 2 0
Sequencing d a t a were compared to the normal LDL receptor c D N A sequence. 2 1
M u t a t i o n analysis
Small deletions ( u p to five nucleotides) are detected by PCR amplification o f the
corresponding fragment followed by agarose gel electrophoresis in a 5% gel. Smaller
rearrangements or single base substitutions are detected by restriction digest
analysis o f the PCR amplification product, in case the nucleotide change creates or
abolishes a restriction site. In all other cases, m u t a t i o n analysis is conducted by
PCR-primer-introduced restriction analysis (PCR-PIRA). Primers required for PCRPIRA are listed in Table 1. Restriction enzyme digest analysis was then conducted
according t o the manufacturer's r e c o m m e n d a t i o n . Discrepancies were resolved by
repeated PCR and analysis o f independent D N A samples.
32
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
Table 1: List of oligonucleotides for PCR-primer-introduced restriction analysis
Name of mutation Location 5' oligonucleotide
Intron 2 5'-TGACAGTTCAATCCTGTCTCTTCAAaG-3'
191 - 2 A - * G
3' oligonucleotide
5'-ACTCCCCAGGACTCAGATAGGC-3'
C163R
Exon4
S'-GTTGGGAGACrTCACACGGTGATGG-S'
S'-CTACTGTCCCCTTGGAACACGTAMGACCCWCC-S1
C201X
Exon4
S'-CAGACGAGGCCTCCTGCCCG-GTGC-S'
S'-CCATACCGCAGTnTCCTCGTCAGATTTGTCCCJ-S1
C234R
Exon 5
5'-GCAATCCTCCTGGCTTGGCCTCCC-3'
5'-CGCTCATGTCCTTGCAGTCATATTCCCGGTCGC-3'
E273K
Exon6
S'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGACGAAACTGAGGCTCAGACACAC-3'
5'-TCTCTAGCCATGTTGCAGACCT-3'
2140+5 G-» A
Intron 14 5'-CGCCCGCCGCGCCCCGCGCCCGGGAC-
S'-GGTACCCATTTGACAGATGAGCAG-S'
ATGAGGAGCTGCCTCACAGGTCT-3'
V776L
a
Exon 16
5'-TCTGTCCATTGTCCTCCCCAGC-3'
5'-GTGATAAAGGACACCGAGTGGGGC-3'
T h e underline represents the m i s m a t c h e d n u c l e o t i d e .
Results
Analysis of frequency and geographical distribution of known FH-causing mutations
in the Dutch population
Restriction enzyme digest analysis o f 13 different point m u t a t i o n s t h a t are k n o w n
to be responsible for FH in the Dutch p o p u l a t i o n was conducted in a c o h o r t o f
1223 heterozygous patients. The type, location and frequency o f the mutations
analysed are summarised in Table 2.
Four m u t a t i o n s were f o u n d w i t h a relatively high frequency. The m o s t c o m m o n
m u t a t i o n s , w i t h a frequency o f 8%, are N 5 4 3 H and 2393del9, which are linked on
the same allele. 1 2 This double m u t a t i o n has also been identified in D e n m a r k 2 2 ,
although w i t h a low frequency.
A splicing mutation in intron 9 , 1 3 5 9 - 1 G —» A, is the second most c o m m o n mutation
with a frequency o f 6.9%. This mutation appears to be frequent also in the
neighbouring country o f Belgium. 2 3
The cluster o f splicing mutations at intron 3, 313 + 1 G —» A and G —» C, 313 +
2 T —* C, represents the third most frequent m u t a t i o n (4.9%). At first, having no
prior indication o f their relative frequency 1 1 , we applied a protocol that allows their
simultaneous detection. However, subsequent analysis tailored to the detection o f
each single defect revealed that all positives for this test were carriers o f the 3 1 3 + 1
G —» A variant. This finding is in agreement w i t h the data on the frequency o f this
m u t a t i o n in other countries. 2 4 , 2 5
Finally, the W 2 3 X m u t a t i o n , occurring w i t h a frequency o f 3 . 1 % , is a recurrent
33
Part 7 Chapter 2
Table 2: Frequency of known mutations
Name
Location Ref.
Detection method
Number o f
carriers (%)
Cohort size
W23X
Exon 2
Stwl+ (PIRA)
38(3.1)
1 223
11
313 + 1 G ^ A
Intron 3
Nde\+ (PIRA)
61 (4.9)
1 223
11
C146X
Exon 4
Dde\ +
5(0.4)
869
11
E207X
Exon 4
Mnll -
21(1.7)
1 223
11
S285L
Exon 6
Msel +
11 (0,9)
1 223
11
R329X
Exon 7
/-/faal-(PIRA)
0
V408M
Exon 9
Nla\\\ +
11 (0,9)
1358+1 G - > A
Intron 9
BstEII-(PIRA)
1
1359-1 G ^ A
Intron 9
Dde\ -
78(6,9)
N543H
Exon 11
Nco\+
98* (8)
1 223
12
P664L
Exon 14
Pst\ +
4(0.32)
1 223
17
2393de19
Exon 17
5% agarose
98*(8)
1 223
12
R350M(Apo13)
Exon 26
Ms/>I-(PIRA)
30 (2.4)
1 223
2
Total
563
11
1 223
44
563
16
1 223
11
358(29.2)
*N543H and 2393d el9 are lin <ed on the same allele.
m u t a t i o n in several European countries and in the United States. 4
D u r i n g the course o f screening, it became apparent t h a t some m u t a t i o n s are rare
a n d , hence, a smaller number of patients were screened.
To evaluate whether a correlation between the frequency and the geographical
d i s t r i b u t i o n o f these m u t a t i o n s can be established, each m u t a t i o n was classified
according to the region o f origin o f the carriers ( d a t a not shown).
Interestingly, the t w o m o s t c o m m o n m u t a t i o n s are most frequently f o u n d in
specific regions
o f the country
(Figure
1). Fifty-four
carriers (69%) o f the
1359-1 G —» A m u t a t i o n originate f r o m the province o f N o r t h - B r a b a n t , in the m i d southern part o f the country, while 65 carriers (67%) o f the N 5 4 3 H - 2 3 9 3 d e l 9
m u t a t i o n originate f r o m the province o f N o r t h - H o l l a n d in the north-west part o f
The Netherlands a n d , more specifically, f r o m the district West-Friesland. The intron
3 m u t a t i o n , 313 + 1 G —» A is more frequently f o u n d in provinces along the eastern
border o f the c o u n t r y (Groningen a n d Gelderland). The W 2 3 X m u t a t i o n in exon 2
is particularly frequent in the northern
provinces (Friesland, Groningen
Drenthe). All other m u t a t i o n s were t o o rare to establish a local preference.
34
and
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
Figure 1 . Geographical distribution of the four common FH-causing mutations
in The Netherlands.
Identification and characterisation o f new mutations
One hundred and forty-one, apparently unrelated, heterozygous FH patients were
screened for sequence alterations in all 1 8 exons o f t h e LDL receptor gene and in the
promoter
region
by DGGE. Prior to
DGGE analysis, the presence o f large
rearrangements(26) and o f t h e R3500Q variant o f t h e A p o B gene(2) was excluded
in this group. Nucleotide alterations were revealed by the presence o f t w o or more
D N A bands on DGGE gels compared w i t h a single band in normal samples and the
underlying molecular defect was then characterised
by direct sequencing. A
m u t a t i o n was identified in 111 patients, yielding a detection rate o f 80%. In
a d d i t i o n to a number o f m u t a t i o n s that have been previously reported in the Dutch
FH p o p u l a t i o n 1 1 " 1 7 , we f o u n d a number o f mutations t h a t were previously not
known in the Dutch p o p u l a t i o n , but that have been f o u n d in other countries
(Table 3). These include the A 2 9 S ( 2 7 ) , the C134G FH-Germany(4), the 1061 - 8
35
Part 1 Chapter 2
( T - > C ) ( 2 8 ) , the E336K FH-Paris 7 ( 4 ) and the V8061 FH-New York 5 ( 4 ) .
However, the vast m a j o r i t y (20 out o f 25) o f the variants do not appear to have been
reported previously in the literature (Table 3). One new polymorphism in exon
4 ( C ^ T a t 6 2 1 , G 1 8 6 G ) is also presented in Table 3.
All m u t a t i o n s and polymorphisms have been identified in only 1 or 2 individuals,
indicating t h a t these variants are rare.
Missense mutations
Twelve missense m u t a t i o n s , which occur most frequently' 4 , were identified. Five o f
t h e m , C 1 1 3 R a n d C163R in exon 4 , C234R in exon 5, C 3 3 1 W a n d C317G in exon
7, involve a cysteine residue, indicating t h a t these m u t a t i o n s are most probably
pathogenic.
The V776L m u t a t i o n is caused by a G —»T transversion at the last nucleotide o f exon
16, which changes the c o d o n for valine at position 776 into a leucine. Therefore, we
classified it as a missense m u t a t i o n . However, this nucleotide is also part o f the
signal sequence required for correct splicing o f the i n t r o n 2 9 and it is most likely that
his m u t a t i o n is responsible for FH also through impairment o f this mechanism.
Splicing mutations
Four new splicing m u t a t i o n s were i d e n t i f i e d . T w o o f t h e m , 1 91 - 2 A ^ * G in intron 2
and 2 3 8 9 + 1 G —» T in intron 16, affect the invariant AG and T dinucleotide o f
acceptor and d o n o r sites, respectively (29). The t h i r d , 67-5del4, is a four nucleotide
deletion which severely disrupts the splicing onsensus sequence at the acceptor site
o f intron 1, including the AG invariant dinucleotide. The f o u r t h splicing m u t a t i o n ,
2140 + 5 G —» A in i n t r o n 1 4 , does not involve nucleotides t h a t are essential or
splicing. However, a G nucleotide at position + 5 o f the 5' d o n o r splice site
consensus sequences is k n o w n to be associated w i t h a consensus value o f 0.84.
Therefore, it is plausible t o assume t h a t the 2140 + 5 G —» A m u t a t i o n is also an FH
causing m u t a t i o n .
Insertions and deletions
W e have reported t w o new deletions in Table 3. One is a five nucleotide deletion in
exon 16, w h i c h results in a frameshiftand premature t e r m i n a t i o n at codon 7 6 5 , just
a few bases d o w n s t r e a m f r o m the deletion. The second one is an in-frame 12 bp
deletion in exon 14, w h i c h deletes four a m i n o acids ( G l n 6 5 7 - Tyr658 - Leu659 Cys660) o f the protein sequence, including a cysteine residue.
The only insertion detected (Table 3 ) consists o f a d u p l i c a t i o n o f 18 nucleotides in
36
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
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Part 7 Chapter 2
exon 17, resulting in the in-frame insertion o f six a m i n o acids (Cys782 - Leu783 Gly784 - Val785 - Phe786 -Leu787) at the end o f the transmembrane d o m a i n o f the
receptor protein.
Discussion
The main objective o f our analysis was to delineate the full spectrum o f m u t a t i o n s
t h a t underlie FH in the D u t c h p o p u l a t i o n , which represents a first step towards the
implementation
o f nation-wide
D N A testing for this disease. T w o
different
approaches were used: first, we estimated the prevalence o f a number o f mutations
t h a t are already k n o w n to be responsible for FH in the D u t c h p o p u l a t i o n a n d ,
second, we searched for additional defects by DGGE screening o f a group o f
patients w i t h an unequivocal clinical diagnosis o f FH.
W e believe t h a t a molecular diagnosis for FH offers several advantages. It is the only
approach t h a t provides a definitive diagnosis, which might become essential before
considering gene therapy. 3 0 Molecular diagnosis is also crucial in family studies,
allowing
identification
o f affected
family members, w h o
require
cholesterol
lowering, while non-affected sibs can be reassured. Identification o f a significant
number o f carriers o f a similar m u t a t i o n will also aid in studying genotypephenotype relationships 3 1 as well as establish the relationship between a given
m u t a t i o n and the response t o lipid-lowering therapy. 3 2 , 3 3
In order t o evaluate the prevalence o f mutations already k n o w n t o be responsible
f o r FH in the Dutch p o p u l a t i o n , we selected 13 mutations t h a t in previous studies
appeared in more t h a n 1 individual. Their frequency was tested in a large c o h o r t o f
FH patients, collected through 4 6 lipid clinics, evenly distributed a m o n g our
provinces. Therefore, we consider these patients as representative o f the overall FH
p o p u l a t i o n in The Netherlands. O u t o f 1223 FH patients, 358 carriers o f the 13
different m u t a t i o n s were identified, representing 29% o f the patients analysed. In
this g r o u p , we identified 30 carriers o f the R3500Q m u t a t i o n in the ApoB gene,
which represents 2.4% o f the cohort examined. These data are consistent w i t h the
frequency reported in other countries. Contrary to the small group o f patients
analyzed for new variants, we did n o t analyze this large c o h o r t o f patients for the
presence o f large rearrangements. Considering t h a t these have been reported w i t h
a frequency o f a b o u t 6% in the Dutch p o p u l a t i o n . 8 ' 9 inclusion o f the large
rearrangements in the screening strategy o f this group w o u l d have increased the
n u m b e r o f carriers identified up to 35%.
38
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
Four LDL receptor gene m u t a t i o n s were detected w i t h a relatively high frequency:
the double m u t a t i o n N 5 4 3 H and 2 3 9 3 d e l 9 , 1 3 5 9 - 1 G - * A, 313 + 1 G - > A and
W 2 3 X . Screening for these four m u t a t i o n s alone w o u l d have resulted in the
identification o f 275 carriers (22.4% o f all patients analyzed). This indicates t h a t an
estimate o f the prevalence o f specific m u t a t i o n s in a given p o p u l a t i o n can facilitate
the selection o f a strategy for the i m p l e m e n t a t i o n o f m u t a t i o n analysis.
W i t h the exception o f a few p o p u l a t i o n s , including the
French-Canadians 3 4 ,
Christian Lebanese 35 , Druze 3 6 , Finns 3 7 and Afrikaner 3 8 , in which a small number o f
m u t a t i o n s explain the majority o f FH cases, in most European countries and in
N o r t h America founder
effects do
not
exist. The
preferential
geographical
d i s t r i b u t i o n o f the t w o most c o m m o n Dutch FH m u t a t i o n s in specific areas o f the
country, Brabant for the 1359 - 1 G - > A and West-Friesland for the double N 5 4 3 H
and 2393de19 m u t a t i o n , represents an unexpected Finding. While the origin o f a
possible founder effect in the mid-southern part o f the country is unclear, the high
prevalence o f the double m u t a t i o n
in West-Friesland
may be attributed
to
geographical isolation. From the 13th century o n w a r d , this region was separated
from the mainland by water and marshes and this situation remained unaltered
until the 17th century, when large areas in N o r t h - H o l l a n d were reclaimed due t o
rapid expansion o f the p o p u l a t i o n . 3 9 Interestingly, the same double m u t a t i o n has
also been recently identified in t w o small FH families in Denmark. 2 2
We also report the identification o f 16 new single base substitutions and four small
deletions and
insertions, w h i c h , added
to
the
point
mutations
and
large
rearrangements previously identified in the LDL receptor gene o f Dutch FH patients,
brings the number o f FH-causing m u t a t i o n s in this country up t o 6 1 . A l t h o u g h the
actual frequency o f the newly identified mutations needs to be evaluated in a larger
group o f patients, none o f them were detected in more than 1 or 2 patients,
suggesting t h a t these mutations are rare.
In general, several arguments suggest t h a t the newly identified m u t a t i o n s are
pathogenic. First, w i t h the only exception o f the 2140 + 5 m u t a t i o n in i n t r o n 14,
these were the only nucleotide changes f o u n d after scanning o f the entire c o d i n g
and splice site consensus sequences o f the LDL receptor gene. Second, these
changes do not occur in the n o r m a l Dutch p o p u l a t i o n , as assessed by DGGE
screening o f 100 control D N A samples. In a d d i t i o n , w i t h the exception o f only one
family in which the 2140 + 5 m u t a t i o n is f o u n d in c o m b i n a t i o n w i t h the E207K
variant, in all other cases family analysis shows a clear pattern o f co-segregation o f
the m u t a t i o n w i t h clinical signs o f F H , such as an elevated LDL cholesterol.
O u t o f the 12 missense m u t a t i o n s , five involve cysteine residues and are m o s t likely
39
Part 1 Chapter 2
t o result in defective protein receptors due t o misfolding. For the remaining seven,
expression o f the m u t a n t genes in m a m m a l i a n cells followed by functional studies
o f the m u t a n t receptors is required to definitively demonstrate their pathogenicity.
In 30 o u t o f the 141 FH patients initially included in the screening strategy for new
LDL receptor defects, we were not able to detect any variant by DGGE, in spite o f
the definite clinical diagnosis, o f the exhaustive screening approach, w i t h the
inclusion o f all exons, promoter region and splice site consensus sequences and o f
the exclusion o f large rearrangements, t h a t cannot be detected by DGGE and o f the
R 3 5 0 0 Q variant in the A p o B gene.
A similar detection rate (-80%) was also o b t a i n e d in a previous study 1 1 , in w h i c h a
smaller g r o u p o f patients was examined. One possible explanation lies in the
detection limit o f the DGGE technique itself, a l t h o u g h DGGE has been proven to be
a powerful detection m e t h o d , it is k n o w n t h a t it is not able to detect all m u t a t i o n s ,
possibly because o f their peculiar position in the fragment or because o f the
s u r r o u n d i n g sequence composition in the fragment t o be analysed. In these cases a
second r o u n d o f screening with a different detection m e t h o d (e.g. single strand
c o n f o r m a t i o n p o l y m o r p h i s m (e.g. [SSCP]), increases the chances o f detecting
nearly all variants. 4 0
A second plausible explanation implies t h a t the m u t a t i o n is located in a gene other
t h a n the LDL receptor. New evidence s u p p o r t i n g this hypothesis has recently been
given by Varret et a!. 3 and implicates a new locus, named FH 3, t h a t might be
responsible for a phenotype indistinguishable f r o m the classical FH according to
the clinical traits.
Considering the large variability of the spectrum o f Dutch FH mutations and the
fact that m o s t m u t a t i o n s are rare, the application o f a molecular diagnostic test for
routine screening o f all mutations requires a strenuous effort.
In this respect, the recent development o f h i g h t h r o u g h p u t methodologies like the
o l i g o n u c l e o t i d e ligation assay (OLA) 4 1 or multiplex allele-specific diagnostic assay
( M A S D A ) 4 2 t h a t enable simultaneous analysis o f a large number o f known
m u t a t i o n s (> 100) in a single assay m i g h t offer a valid alternative to the laborious
and time c o n s u m i n g assays for specific m u t a t i o n s .
A l t h o u g h the n u m b e r o f D u t c h FH m u t a t i o n s identified is high, the database o f all
FH-causing m u t a t i o n s in the Dutch p o p u l a t i o n is far from complete. It has been
estimated t h a t there are a b o u t 4000O FH heterozygotes in the Dutch p o p u l a t i o n . 4 3
Considering t h a t so far in this as well as in previous studies only a limited number
o f patients have been scanned for u n k n o w n defects and t h a t most m u t a t i o n s are
present in single families, the actual n u m b e r o f D u t c h FH m u t a t i o n s may be much
40
MOLECULAR GENETIC TESTING FOR FAMILIAL HYPERCHOLESTEROLEMIA
higher. Therefore, predictions on the total number o f LDL receptor
defects
accounting for FH in our p o p u l a t i o n cannot be made a n d , as a consequence,
estimates o f the predictive value o f a multiplex testing p r o t o c o l remain inaccurate.
C o n t i n u a t i o n o f this approach may increase the number o f LDL receptor mutations
t o several hundreds. For this reason, screening for new m u t a t i o n s by DGGE remains
a major objective o f our laboratory.
In a d d i t i o n , strict selection criteria were applied t o the patients included in this
study. Therefore, mutations leading t o a mild phenotype o f FH might have been
missed. A multiplex testing protocol w o u l d therefore result in a relatively low
predictive value, inherent to the selection criteria applied.
In summary, w i t h the w o r k described in this paper we have obtained more insight
into the molecular basis o f FH in The Netherlands. Sixty-one different m u t a t i o n s
have been f o u n d
to
be responsible for the disease:these
rearrangements, reported elsewhere
8,9
include ten
large
and 51 p o i n t m u t a t i o n s . O u t o f these, 20
are novel m u t a t i o n s . The vast majority o f m u t a t i o n s have been found in 1 or 2
individuals. Only four mutations present w i t h a relatively high frequency a n d ,
altogether, account for 22% o f all patients examined.
Acknowledgements
This study was financially supported
by Praeventiefonds ( # 2 8 - 2 4 0 3 ) . Dr JJP
Kastelein is a clinical investigator o f The Netherland
Heart Foundation. The
technical assistance o f Laura Kerkhof is gratefully acknowledged.
41
Part 7 Chapter 2
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