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SUPPLEMENTARY MATERIAL AND METHODS
Patients and controls
A total of 73 unrelated patients diagnosed with knee or hip OA were
included in the present study. Twenty-five of these patients carried haplogroup
J, 25 haplogroup U and 23 haplogroup H. The patients meeting the inclusion
criteria for this study included 52 females and 21 males (mean age: 67.74 ±
8.96 years; range: 51-95), who had been diagnosed with OA according to
American College of Rheumatology (ACR) criteria. Knee and hip radiographs
from 148 subjects were classified according to Kellgren and Lawrence (K/L)
scoring from Grade I to Grade IV. The 77 healthy controls who met the inclusion
criteria for normal subjects included 39 females and 38 males (mean age: 66.01
± 11.88; range: 42-94), not having knee or hip OA according to the ACR criteria.
Twenty-five of the healthy controls carried haplogroup J, 25 haplogroup U and
27 haplogroup H. Clinical data including gender, age and smoking status were
collected for all the subjects. In all cases, informed consent and the agreement
of the ethical committee from the Galician Health Administration were obtained.
Single Base Extension (SBE) assay
SBE assay consists in the annealing of a single primer to a sequence of
the mtDNA template that contains the SNP we want to interrogate, such that the
3´-end of this primer falls one base short of the SNP site present on the
template. Since we are using only dideoxynucleotides (ddNTPs) in the reaction,
when the complementary base is incorporated by the Taq DNA polymerase, the
elongation stops and, depending on the fluorescence emitted, the SNP site will
be identified. In contrast, those minor samples that did not belong to any of
these 6 mtDNA haplogroups, were analyzed by PCR-RFLP.
Six specific primers are designed in order to amplify the mtDNA fragments that
contain each of the informative SNPs, which characterize the 6 major European
mtDNA haplogroups, in one multiplex reaction. The polymorphic sites analyzed
(7025, 14766, 10394, 4577, 12308 and 4216) were previously reported.
Besides, another 6 specific SBE primers were also designed to interrogate each
SNP site. In order to design the SBE primers, several considerations were
taken into account: (i) the minimum length of each primer consisted of 20 nt, (ii)
each primer had a Tm of ~60ºC and (iii) the minimum difference in length
among the 6 primers was of 6nt in order to avoid overlapping. In this latest
case, is possible to add unspecific nucleotides at 5´-end of the primer for
increasing its length. The sequence of PCR, PCR-RFLP and SBE primers are
avialabe in Rego et al 2008 [1].
Multiplex PCR mixture consisted of a final concentration of 1X Reaction Buffer
(Bioline), 0.2mM of each deoxynucleotide (dNTP) (Bioline), 1.5mM MgCl 2
(Bioline), 0.025 U/µL of BioTaq DNA polymerase (Bioline) and 0.3 µM of each
primer in a volume of 50 µL. Blood isolated (Invisorb Spin Blood Mini Kit from
Invitek) DNA (75 ng) was added to the mixture and amplified as follows: 94ºC
for 5 minutes, 35 cycles at 95ºC for 60 seconds, 55ºC for 60 seconds and 72ºC
for 60 seconds, and a final extension at 70ºC for 10 minutes. After amplification,
the PCR products were optionally electrophoresed in a agarose gel and uvvisualized after ethidium bromide treatment.
To remove primers and unincorporated dNTPs, multiplex PCR products were
treated
with
ExoSap-IT
(Amersham)
following
manufacturer´s
recommendations: adding 2µL of enzyme to 5 µL of PCR product (or a
proportional ratio), followed by an activation of the enzyme at 37ºC for 15
minutes and a further deactivation by incubating at 80ºC for 15 minutes. Later,
the samples were placed on ice.
Multiplex SBE reactions were carried out following the recommendations from
Applied Biosystems with some modifications. So, in a final volume of 10µL, we
added 1.5µL of SNaPshot® Multiplex Kit, 2.5µL of purified PCR product and a
final concentration of 0.2µM of the SBE primers mixture. To reach the final
volume of 10µL, ddH2O was added. Thermal cycling conditions for SBE were as
follows: 96ºC for 60 seconds and 25 cycles at 96ºC for 10 seconds, 60ºC for 5
seconds and 60ºC for 30 seconds. To remove unincorporated ddNTPs, the SBE
reaction products were treated with Shrimp Alkaline Phosphatase (SAP)
(Amserham) by adding to the mixture 1µL of enzyme, 2µL of SAP reaction
buffer and 7µL ddH2O, followed by an activation of the enzyme at 37ºC for 1
hour and a further deactivation by incubating at 75ºC for 15 minutes.
Subsequently, the samples were placed on ice.
Finally, 9µL of Hi-DiTM Formamide (Applied Biosystems), 0.5µL of size internal
standard (120 Liz Size Standard from Applied Biosystems) and 0.5µL of purified
SBE product were mixed and denatured at 95ºC for 5 minutes prior to load into
an ABI 3100 genetic analyzer. The configuration system is based on a 36cm
length capillary filled with a Performance Optimized Polymer 4 (POP4)
containing urea, from Applied Biosystems. The SBE default run module
consisted in 22 seconds of injection time, 16 minutes of run time and a run
voltage of 15 KVolts. Once the runs were finished, the whole data was analyzed
in GeneMapper v3.5 software (Applied Biosystems), which is able to assign the
different alleles (SNPs) in each locus, prior designing of a reference sequence
that encompasses all the allelic variants for each locus . In order to verify the
results obtained, some samples were analyzed by PCR-RFLP and direct
sequencing of the PCR product.
Determination of serum levels of molecular biomarkers
Coll2-1, Coll2-1NO2, C2C and CPII are markers for cartilage metabolism,
HA marks synovial metabolism and YKL-40 is a marker for both cartilage and
synovium turnover. Determination of Coll2-1 and Coll2-1NO2 was performed at
the University of Liege (Belgium), at the Bone and Cartilage Research Unit,
following the below described assay. The remaining biomarkers were measured
in our facilities using enzyme-linked immunosorbent assays (ELISA) and
following the manufacturers’ recommendations. Serum C2C and CPII were
measured using kits from Ibex Technologies (Montreal, Quebec, Canada), HA
was measured using a kit from Corgenix Medical Corporation (Denver, CO,
USA) and YKL-40 was measured using a kit from Quidel Corporation (San
Diego, CA, USA).
The determination was performed by mixing in the same run OA patients
and healthy controls, regardless of their mtDNA haplogroup.
Coll2-1 and Coll2-1NO2 concentrations were measured by two
competitive and specific immunoassays (ELISA) [2]. The Coll2-1 immunoassay
only measured the amino acid sequence 108HRGYPGLDG116 in its linear form
while the Coll2-1NO2 immunoassay quantified with a high specificity and affinity
the nitrated amino acids sequence. The limits of detection were 17 nM for Coll21 immunoassay and 25 pM for Coll2-1NO2 immunoassay. The intra- and interassays coefficients of variation were lower than 10 % and the dilution curves
were parallel to the standard curve for both assays. The analytical recoveries
were in mean 104.7 % and 121.9 % for Coll2-1 and Coll2-1NO2 assays,
respectively.
Briefly, microplates were coated with 200 µl of streptavidine 0.5 mg/ml
during 48 hours. After washing (buffer washing: Tris 25 mM, NaCl 50 mM,
Tween 20 0,2% (v/v) pH 7.3), microtiter plates were subsequently blocked with
400 µl/well of blocking buffer (KH2PO4 1.5 mM, Na2HPO4 8 mM, KCl 2 mM,
NaCl 138 mM, BSA 0.5 % (v/v) pH 7.2) overnight at 4°C. Coll2-1 and Coll21NO2 were conjugated to biotin according to the method described by
Rosenquist et al [3]. After washing, 100 µl of these biotinylated peptides either
Coll2-1 at 2.5 ng/ml or Coll2-1NO2 at 1.25 ng/ml, were added to each well of
streptavidine-coated plates and incubated for two hours at room temperature.
After washing, 50 µl of calibrators (synthetic peptide) or unknown samples,
diluted in incubation buffer (10 mM phosphate buffer saline (PBS), 138 mM
NaCl, 7 % (w/v) BSA, 0.1 % (v/v) Tween 20 pH 7.0 for the Coll2-1
immunoassay and in 50 mM Tris, 138 mM NaCl, 7 % (w/v) BSA, 0.1% (v/v)
Tween 20 pH 8.0 for the Coll2-1NO2 immunoassay), were added to the wells,
followed by either 100 µl of D3 antibody (for Coll2-1), diluted 1/40,000 or 100 µl
of D37 antibody (for Coll2-1NO2), diluted 1/500,000, and incubated one hour at
room temperature. The dilutions of the antisera and of the secondary antibody
were done in buffer dilution [10 mM PBS, 138 mM NaCl, 0.2% (w/v) BSA, 0.1%
(v/v) Tween 20 pH 7.0 for the Coll2-1 immunoassay and in 50 mM Tris, 138 mM
NaCl, 0.2% (v/v) BSA, 0.1% (v/v) Tween 20 pH 8.0 for the Coll 2-1NO2
immunoassay]. After washing, 100 µl of peroxidase-conjugated goat antibodies
to rabbit IgG (Biosource, Belgium), diluted 1/5,000 in incubation buffer, were
incubated one hour at room temperature. After washing, 100 µl of freshly
prepared enzyme substrate (TMB, Biosource, Belgium) were added into each
well. After 15 minutes, the reaction was stopped with 100 µl of 4M H 3PO4. The
coloration was read with a microplate reader (Labsystem, Finland) at 450 nm,
corrected for absorbance at 650 nm.
Statistical analysis
The Coll2-1NO2/Coll2-1 ratio was utilized as an additional measure of
oxidative stress-mediated cartilage degradation, and the C2C/CPII ratio as a
further index of cartilage breakdown.
As a first approach, a non-parametric study was performed. The MannWhitney U-test was used to compare serum molecular marker concentrations
between OA patients and healthy controls, and the Kruskal-Wallis test was used
to compare the molecular marker concentrations among the three different
haplogroups (J, U and H), regardless of diagnosis. As a second approach, a
multiple regression analysis (MRA) was performed to evaluate the influence of
gender, age, smoking status, haplogroup, diagnosis and possible interactions
between haplogroup (J, U or H) and diagnosis (OA patient or healthy control) on
serum levels of the molecular markers (dependent variable). A distribution
analysis using the Kolmogorov-Smirnov test showed that most of the molecular
markers were not normally distributed; therefore, concentrations of markers
were log, square root or inverse transformed to obtain a normal distribution for
this approach. When appropiate, the Bonferroni post hoc multiple comparisons
test was also performed.
The influence of radiographic grade to the serum levels of the molecular
markers was tested using both the non-parametric Jonckheere-Terpstra test for
ordered groups and MRA. For this approach we obtained, as indicated above,
the radiological grade of 148 subjects out of the 150 that are part of the study.
Using K/L scores, subjects were divided into three radiographic groups: group A
included 77 healthy controls (K/L grade 0 and grade I), group B consisted of 47
OA patients with K/L grades II and III, and group C included 24 OA patients with
K/L grade IV.
References (not cited in the main manuscript)
1.
Rego I, Fernández-Moreno M, Relaño S, et al. The use of single base
extension (SBE) for the assessment of European mitochondrial DNA
haplogroups in Galician population. The open cell development &
biology journal. 2008;1:10-16
2.
Deberg M, Labasse A, Christgau S, et al. New serum biochemical
markers (Coll 2-1 and Coll 2-1 NO2) for studying oxidative-related
type II collagen network degradation in patients with osteoarthritis and
rheumatoid arthritis. Osteoarthritis Cartilage 2005; 13: 258-265.
3.
Rosenquist C, Fledelius C, Christgau S, et al. Serum CrossLaps One
Step ELISA. First application of monoclonal antibodies for
measurement in serum of bone-related degradation products from Cterminal telopeptides of type I collagen. Clin Chem 1998; 44: 22812289.