2
.
8
. Binding, uptake and degradation of
125
I-labeled lipoproteins
VLDL d B 1.0063 kgl and LDL 1.019 B d B 1.063 kgl were isolated by preparative ultracentrifugation
from a pool of normolipemic donors and iodinated using the iodine monochloride method [30]. Human
skin fibroblasts were obtained from skin biopsies of the patient and a normolipemic individual, respectively.
Cells were grown in 24-well polystyrene plates. Prior to the experiments, cells were pre-incubated for 40 h in
medium containing 10 vv human lipoprotein-defi- cient serum in order to up-regulate LDL receptors.
Binding, uptake and degradation of
125
I-labeled lipo- proteins were measured according to the procedure
described by Goldstein et al. [31] with slight modifica- tions [32]. To measure cell surface binding, cells were
incubated for 1 h at 4°C with
125
I-labeled lipoproteins in DMEM medium containing 10 mM HEPES. To
determine uptake surface binding plus internalization and degradation, cells were incubated for 4 h at 37°C
with
125
I-labeled lipoproteins in DMEM medium con- taining 24 mM bicarbonate pH 7.4. The amount of
125
I-labeled material associated with the cells binding and internalization was determined as
125
I-labeled trichloracetic acid-soluble non-iodine material in the
conditioned medium. Values were corrected for protein concentrations using the Lowry assay and BSA as
standards Bio-Rad, Hercules, CA.
3. Results
3
.
1
. Family analysis The pedigree of the G. family is shown in Fig. 1 and
the plasma lipid values are given in Table 1. The parents of the patient were first degree cousins,
as the parents’ mothers were sisters PID III-2 and -3. Further, the maternal grandparents of the patient were
also first degree cousins PID III-1 and -2, so that an extraordinary extent of consanguinity is evident in this
family. The patient showed lipid values that definitively met the biochemical criteria for homozygous FH. The
patient presented with xanthomas on the proximal in- terphalangeal joints of the fingers and bilateral lipoid
arcs characteristic of FH. On ECG, discrete repolarisa- tion abnormalities were detected, which we interpret as
an early sign of ischemic cardiomyopathy. The patient is now treated with colestyramine 16 gd and LDL-
apheresis which is performed every 2 weeks.
Both siblings of the patient also showed concentra- tions of LDL-chol that were above the 95th percentile
for age and gender in the German population. Both siblings were doing well and had no clinical signs of
FH. The
mother showed
moderate hypercholes-
terolemia only with LDL-chol and HDL-chol concen- trations that were both above the 75th percentile for
age and sex. Fasting triglycerides, however, were signifi- cantly elevated.
Of special interest was the biochemical analysis of the patient’s father, who showed an extreme fasting hyper-
trigyceridemia and an unusually high VLDL-chol con- centration. The concentration of LDL-chol, however,
was 70 mgdl, thus being below the 5th percentile of age — and sex adjusted reference values. Consequently, at
a glance, the presence of a mutated LDL-R allele appeared to be rather unlikely in this individual. Both
parents showed no clinical signs of FH. The self-re- ported alcohol consumption of the parents was 150 and
600 g ethanol per week for mother and father, respec- tively. The g-glutamyl-transferase activity of the father
was elevated at 87 Ul local reference range: 0 – 24 Ul.
Unfortunately, because the patient’s parents have completely lost contact with their relatives, further
members of the G. family were not available for clinical examination, DNA testing, or biochemical analyses.
Information on the causes of death in previous genera- tions was also not obtainable.
3
.
2
. Identification of a sequence alteration by DGGE Southern blot analysis after BglII or XbaI digestion
of genomic DNA isolated from the patient, his parents and his siblings did not reveal any major abnormality
in the LDL-R gene not shown, suggesting the pres- ence of a point mutation or minor rearrangement. To
determine the exact location of the mutation, the am- plified products of all 18 exons, including the splice site
sequences, and of the promoter of the LDL-R of the patient, his parents and his siblings were subjected to
DGGE analysis.
As shown in Fig. 2, evidence for a single mutation was found in exon 7 or in its splice junctions.
The DGGE analysis for the patient revealed a single homoduplex band as it is seen in individuals with two
identical alleles. However, the electrophoretic mobility of this homoduplex band was clearly distinct from that
of two normolipemic controls. The mutant homoduplex band migrated a shorter distance in the denaturing
gradient gel, indicating a lower melting temperature of the altered sequence. The appearance of this single
homoduplex band with an aberrant melting profile clearly indicated a true homozygous state with the
presence of an identical mutation on both alleles.
In contrast, in the DGGE analysis of the patient’s parents and his siblings an identical four band pattern
was apparent, indicating the presence of two different alleles of the LDL-R gene. While the two lower bands
were caused by the two different alleles, the remaining two bands were caused by formation of heteroduplexes
between the two alleles. The lowest band migrated at
M .S
. Nauck
et al
. Atherosclerosis
151 2000
525 –
534
529 Table 1
Biochemical data on members of the G. family with the C317Y mutation of the LDL-R gene
a
TG LDL-chol
Chol HDL-chol
Age VLDL-chol
Sex C317Y
Apo E PID
Apo B Lpa
Apo AI mgdl
mgdl mmoll
mgdl mmoll
mmoll mmoll
mmoll 2.1
IV 1 1.0
F Heteroz.
24 139
227 110
47 7.3
2.8 4.2
1.1 6.5
IV 2 Heteroz.
M 23
119 179
3 37
9.4 21.3
1.8 1.1
0.8 Heteroz.
34 164
129 6.2
53 8.1
1.8 V 1
F 14
25.9 M
1.0 0.9
Homoz. 24
564 110
219 9
27.8 1.9
V 2 0.9
5.6 1.3
0.1 Heteroz.
23 126
128 65
V 3 M
11 7.1
a
PID, pedigree identification number; F, female; M, male; ApoE, Apo E phenotype; age refers to years of age at blood sampling; lipid values are in mgdl and represent maximal values obtained before any medical treatment.
Fig. 2. DGGE analysis of the patient and his family. Exon 7 was amplified from genomic DNA as described in Section 2. The PCR
product, containing a 40-bp GC clamp was loaded on a 6 poly- acryl-amide gel containing a denaturing gradient of 40 – 80. Pedigree
identification number is with reference to Table 1. A single homodu- plex band with an abnormal electrophoretic mobility, indicating the
presence of an identical mutation on both alleles is seen in subject V2. All other family members examined display an identical four band
pattern with a fast-migrating homoduplex band at the position of the normolipemic controls lane 6 and 7 and a slow-migrating homodu-
plex band at the position corresponding to the mutant DNA.
bonds that are located in repeat A of the EGF precur- sor homology domain of the LDL-R protein [2].
As the base substitution at position 1013 creates a recognition sequence of RsaI GTAC, the presence of
the mutation was confirmed by RsaI digestion of the PCR product of exon 7. As expected, the 253 bp PCR
product originating from the patient was completely digested, generating two fragments with 146 and 107
bp, respectively. The PCR products of the controls remained completely uncut, whereas the other family
members displayed restriction fragments consistent with a heterozygous state for this mutation not shown.
3
.
4
. Haplotype analysis Genotyping at seven polymorphic sites showed that
the index patient was homozygous for the SfaNI + , StuI + , AciI + , HincII − , A6aII + , MspI
+ and the NcoI − RFLPs. This genotype was identical for all family members except for the NcoI
polymorphic site, at which the mother and the sister of the patient were heterozygous.
3
.
5
. Interaction of lipoproteins with cultured cells To examine the functional properties of the mutant
LDL-R, skin biopsies of the patient and a nor- molipemic individual were obtained and primary
fibroblast cell cultures were established. We studied binding at 4°C, uptake and degradation both at
37°C of
125
I-LDL and
125
I-VLDL at protein concentra- tions of 5 through 40 mgl. Binding, uptake and degra-
dation of
125
I-LDL in fibroblasts from the patient were less than 10 of those in normal cells Fig. 3. The
interaction of
125
I-VLDL with cultured cells was also decreased, but to a lesser extent. On average, binding,
uptake and degradation of VLDL in cells from the patient were reduced by 60, 30, and 38, respectively,
compared to normal cells Fig. 4. the same position as the homoduplex band of the
controls, representing the normal allele whereas the second lowest band displayed the same elctrophoretic
mobility as the homoduplex band seen in the patient’s sample.
3
.
3
. Nucleotide sequence of the mutant allele and restriction genotyping of the C
317
Y mutation To precisely identify the mutation in exon 7, the PCR
product originating from the mutant allele of the pa- tient was sequenced. Direct bidirectional sequencing
revealed a single base substitution G A at nucle- otide 1013 of the LDL-R cDNA not shown. This
mutation results in the substitution of cysteine by ty- rosine at residue 317 thereby eliminating the fourth of
the six cysteine residues involved in the disulphide
Fig. 3. Binding, uptake and degradation of
125
I-LDL. LDL were prepared by ultracentrifugation and labelled with
125
I as described in Section 2. Skin fibroblasts were grown in DMEM with 10 vv fetal calf serum FCS. Prior to the experiment 40 h, the cells were switched to medium
containing 10 vv human lipoprotein-deficient serum. Cells from the patient rhombs and from a normolipemic donor squares then received
125
I-labeled LDL. Binding panel A, uptake panel B, and degradation panel C were determined as described in Section 2. Each data point represents the average of two experiments, each performed in triplicate.
Fig. 4. Binding, uptake and degradation of
125
I-VLDL. VLDL were prepared by ultracentrifugation and labelled with
125
I as described in Section 2. Skin fibroblasts were grown in DMEM with 10 vv FCS. Prior to the experiment 40 h, they were switched to medium containing 10 vv
human lipoprotein-deficient serum. Cells from the patient rhombs and from a normolipemic donor squares then received
125
I-labeled VLDL. Binding panel A, uptake panel B, and degradation panel C were determined as described Section 2. Each data point represents the average
of two experiments, each performed in triplicate.
Binding of VLDL was also determined after down- regulating the expression of the LDL-R gene by incu-
bating the cells for 24 h in the presence of sterols [33]. In the cells of the patient, sterol-dependent repression
of the LDL-R gene diminished the residual binding activity for VLDL by 50 Fig. 5.
4. Discussion
