Detection of LDL receptor mutations by PCR, PCR-RFLP and Southernanalysis of DNA from Oragene®/saliva samples†
L. Boulet‡, P. Lem** and L. Bernier‡‡ Clinical Research Institute of Montreal, Montreal, QC, Canada** DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

The Oragene® self-collection kit is a non-invasive
method of collecting DNA from saliva and it
gives the same results as DNA from blood forthe analysis of mutations in the LDL receptor
gene by PCR, PCR-RFLP and Southern analysis.

to the prepIT™•L2P protocol7. Additional DNA
was purified from 10 mL of whole blood using theQIAamp® DNA blood maxi kit (Qiagen). All of thesamples were tested for five mutations: >15 kb and5 kb deletions, and mutations in exons 3, 4, and 14.

Introduction>15 kb and 5 kb deletions

Familial hypercholesterolemia (FH) is an autosomal
dominant disorder that occurs about one in 500
individuals in Western populations. It is primarily
caused by mutations in the LDL receptor (LDLR)
gene and over 770 mutations have been describedworldwide1. Among French Canadians, the prevalenceof FH heterozygotes has been estimated to be aboutone in 2702. Due to a genetic founder effect, fivemutations in the LDLR gene account for approximately
83% of FH diagnosed in French Canadians3. Thesemutations are gene deletions of >15 kb and 5 kb, andthree missense mutations: Trp66 → Gly (exon 3), Glu207→ Lys (exon 4), and Cys646 → Tyr (exon 14). Methods
such as Southern blotting4, PCR-RFLP5, and PCR6
have been used to detect these mutations in DNA fromblood. Unlike blood, the Oragene self-collection kit
is a non-invasive method for collecting DNA fromsaliva. The purpose of this study was to compare thedetection of these five LDLR gene mutations in DNA
from blood and DNA from Oragene/saliva samples.

The >15 kb and 5 kb deletions in the LDLR gene weredetected using two methods. First, 10 µg of genomic
DNA from blood and Oragene were digested with therestriction enzymes XbaI and EcoRV, and analyzed
with Southern blotting using the method of Ma et al.
(1989). Second, the PCR assays described by Simard
et al. (2004) were also used to test for both deletions.

Materials and methods
DNA collection and purification
Blood and saliva samples were collected from nine
healthy French-Canadian volunteers and five patients
who had been previously diagnosed with FH due tovarious mutations in the LDLR gene. Saliva wascollected using the Oragene self-collection kit(DNA Genotek) and DNA was purified according†

Exon 3 and exon 4 mutations
Exons 3 and 4 were amplified by PCR, digested with
BslI and MnlI respectively, and electrophoresed on
2.5% MetaPhor agarose gels (Cambrex) according
to the method of Minnich et al. (1995).

Exon 14 mutation
Following the method of Vohl et al. (1995), mismatchprimers were used to create a NlaIII restriction site in
the exon 14 mutant but not in the normal allele. After
digestion, the mutant allele gave two fragments of
163 and 33 bp in size, while the normal allele resulted
in a 196 bp fragment. The fragments were separated
by electrophoresis on 2.5% MetaPhor agarose gels.

Results
DNA yield
The average DNA yield was 159 µg from 2 mL ofsaliva collected with Oragene kits. The average
A260/280 ratio was 1.94.

Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

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Mutation detection

MNHTZ

1

2

3

4

5

6

7Table 1 summarizes the results of testing for thefive mutations by PCR, PCR-RFLP, and Southern
analysis. Figure 1 shows a sample result for PCRRFLP analysis of the mutation in exon 3.
Patient #Oragene results

Blood results

1--

2--

3-

-

4--

Discussion and conclusions

5--

6--

7--

8--

9--

10-

-11>15 kb>15 kb12>15 kb

>15 kb13

exon 14exon 1414exon 3exon 3

Patients #1 to 9 were healthy volunteers with no signs
or symptoms of FH. As expected, they all tested
negative for the five mutations using DNA fromblood and Oragene. Patient #10 had the clinicalcharacteristics of FH, but had previously tested
negative for the five mutations with DNA from blood.
The same negative result was seen again with DNA
from Oragene/saliva samples. Patients #11 to 14 hadbeen diagnosed with LDLR mutations with DNA
from blood and the same results were seen withDNA from Oragene/saliva samples. Overall, sample
collection was easier and non-invasive with Oragene,and the PCR, PCR-RFLP, and Southern blot results
were the same as with DNA from blood.

Figure 1: PCR-RFLP results for detection of the LDLR gene mutation inexon 3. Lanes 1 and 2 show results with DNA from blood for Patients#1 and 2. Lanes 3 to 7 show results with DNA from Oragene forPatients #1 to 5. (M=Marker, N=Normal, HTZ=Heterozygote).

Table 1: Comparison of results from blood and Oragene for detectionof >15 kb and 5 kb deletions, and mutations in exons 3, 4, and 14 of
the LDLR gene.

References
1234567Heath, K., Gahan, M., Whittall, R. and Humphries, S. (2001). Low-density lipoprotein receptor gene (LDLR) world-wide website infamilial hypercholesterolaemia: update, new features and mutation analysis. Atherosclerosis. 154: 243–246.
Moorjani, S., et al. (1989). Homozygous familial hypercholesterolemia among French Canadians in Quebec province. Arteriosclerosis.
9: 211–216.
Minnich, A., Roy, M., Chamberland, A., Lavigne, J. and Davignon, J. (1995). New methods for rapid detection of low-density
lipoprotein receptor and apolipoprotein B gene mutations causing familial hypercholesterolemia. Clinical Biochemistry. 28(3): 277-284.
Ma, Y., Betard, C., Roy, M., Davignon, J. and Kessling, A. (1989). Identification of a second “French Canadian” LDL receptor genedeletion and development of a rapid method to detect both deletions. Clinical Genetics. 36(4): 219-28.
Vohl, et al. (1995). Rapid restriction fragment analysis for screening four point mutations of the low-density lipoprotein receptor genein French Canadians. Human Mutation. 6(3):243-6.
Simard, L., et al. (2004). The Δ>15Kb deletion French Canadian founder mutation in familial hypercholesterolemia: rapid polymerase
chain reaction-based diagnostic assay and prevalence in Quebec. Clinical Genetics. 65: 202-208.
Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.

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