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Technical Briefs |
1
Renal Transplant Unit,
2
Nephrology Department, and
3
Hormonal Laboratory, Hospital Clinic, University of Barcelona, Institut d'Investigació Biomediques August Pi i Sunyer, 08036 Barcelona, Spain;
a address correspondence to this author at: Renal Transplant Unit, Hospital Clinic, 170 Villarroel, 08036 Barcelona, Spain: fax 34-93-2275498, e-mail jmcampis{at}medicina.ub.es
Transforming growth factor-ß1 (TGF-ß1) modulates a wide spectrum of biological activities, including cell proliferation, differentiation, and adhesion as well as extracellular matrix formation. The involvement of TGF-ß1 in inflammation and tissue repair has been established in the past few years, suggesting a crucial role for this cytokine in pathologies of the cardiovascular system, kidney (chronic allograft rejection in kidney transplantation, glomerulonephritis, and diabetic nephropathy), bones (osteoporosis), skin (systemic sclerosis), lung (idiopathic lung fibrosis and asthma), and liver (cirrhosis).
The human TGF-ß1 gene (TGFB1), located on chromosome 19q13, contains seven exons that give rise to a precursor protein of 390 amino acids (1), which is proteolytically processed to generate the mature protein of 112 amino acids. The promoter for TGF-ß1 contains two major sites for the initiation of transcription and multiple regulatory motifs, but it lacks the typical TATA or CAAT boxes (2).
Seven TGFB1 polymorphisms recently have been reported as single-stranded conformational polymorphisms and applied to a multicenter study population, the Etude Cas-Témoin de L'Infarctus du Myocarde (ECTIM) study (3). Three of the seven allelic variations were localized in the 5'-flanking region of the TGFB1 gene (at positions -988, -800, and -509), three were in the coding region (codons 10, 25, and 263), and an insertion was also described in the 5'-untranslated region at position +72.
The analysis of candidate genes and/or polymorphisms has been widely applied in clinical genetics to study human diseases. A rapid and easy method for genotyping is essential in such studies, in which large numbers of cases and controls are needed. The ECTIM study used allele-specific oligonucleotides (ASOs). After amplification by PCR, products were blotted into a nylon membrane and hybridized with an ASO probe. The major disadvantage of this method is that it its time-consuming and requires radioactively labeled oligonucleotides.
We describe here a new, rapid, nonradioactive, and relatively inexpensive method for TGFB1 genotyping, using restriction site polymorphism (RSP) methodology. A fragment of the promoter or coding region of the TGFB1 gene is amplified by PCR and subsequently digested with restriction enzymes: MaeIII, AocI, MspA1I, and FseI for the allelic variants located at positions -800, -509, codon 10, and codon 25, respectively.
We studied DNA from 73 cadaver organ donors from our Institution with no associated medical disease (e.g., arterial hypertension, any type of nephropathy, chronic obstructive pulmonary diseases, and chronic liver diseases) and no chronic diseases. Blood samples (10 mL) were collected into EDTA tubes and used for the separation of white cells. Genomic DNA was purified from peripheral leukocytes by salting out (4).
Published sequence information of the TGF-ß1
promoter gene (2) was used to design a pair of primers
spanning positions -800 and -590; this amplification will
be referred to as F1-F2. Primers for codons 10 and 25 were kindly
provided by I. Hutchinson (University of Manchester, UK), and this
amplification will be referred to as F3-F4. The primer positions,
sequences, and product sizes for the F1-F2 and F3-F4 amplifications are
listed in Table 1
, and a schematic showing the positions of both polymorphic and
nonpolymorphic restriction sites is shown in Fig. 1
A.
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PCR conditions were the same for both amplifications: 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1 µmol/L each primer, 0.2 mmol/L each dNTP, 25 ng of genomic DNA, 2.0 U of Taq DNA polymerase (Boehringer Mannheim), and 50 mL/L formamide in a 30-µL reaction mixture. PCR contamination was checked by the inclusion of negative controls. PCR reactions were initially denatured at 94 °C for 5 min and subjected to 35 cycles in a PTC-100 thermal cycler (MJ Research) with 30 s of denaturing at 94 °C, 30 s of annealing at 60 °C, and 30 s of extension at 72 °C. A final elongation step of 5 min at 72 °C was also included. The identities of the F1-F2 and F3-F4 amplification products were confirmed by restriction enzyme digestion (data not shown).
Amplified DNA (5–10 µL) was digested in a 20-µL reaction mixture, using the buffers and temperatures recommended by the manufacturers. The enzymes used were as follows: MaeIII and AocI (Boehringer Mannheim), MspA1I and FseI (New England Biolabs). Digestions were carried out for at least 2 h. The amount of restriction enzyme required for each digestion was as follows: 1.5 U for MaeIII, 5.0 U for AocI, 10.0 U for MspA1I, and 2.0 U for FseI. Digested products were fractionated in 7.0% polyacrylamide gels and visualized by ethidium bromide staining and transillumination with ultraviolet light. The size marker was pBR322 cleaved with HaeIII (DNA molecular size marker V; Boehringer Mannheim).
Hardy-Weinberg equilibrium was tested by 
2
with one degree of freedom. Comparisons of genotype frequencies between
the ECTIM control group and the present results were performed by
2 with two degrees of freedom. In cases
involving RSPs with low frequencies, groups were combined and subjected
to the Fisher exact test.
Results for the AocI and MaeIII digestion for the
5'-end RSPs of the TGFB1 gene are shown in Fig. 1B
. The
G
A transition at position -800 was detected by the loss of the
restriction site for MaeIII: the G allele
is defined by the presence of two polymorphic bands of 205 and 137 bp,
whereas the A allele is defined by the absence of the
MaeIII site and is detected by a 342-bp band.
MaeIII digestion of the F1-F2 amplification product produces
a constant 114-bp fragment.
The RSP located at position -509 was described as a CT transition.
In this case, the C allele was detected by the presence of
429- and 26-bp bands, whereas the T allele is defined by the
loss of the AocI site, yielding an undigested 455-bp
fragment.
Band patterns for codons 10 and 25 are shown in Fig. 1B
. A TC
transition at codon 10 produces the Leu10Pro
substitution in the signal peptide of the
TGF-ß1 precursor protein and is detected by the
MspA1I restriction enzyme. MspA1I restriction
digestion of F3-F4 amplification produces four constant fragments of
67, 67, 41, and 40 bp. The Pro allele is defined by the presence of
polymorphic bands of 273 and 12 bp, and the Leu allele is defined by a
285-bp fragment.
The Arg25Pro polymorphism, a GC
transversion, was detected by the loss of the FseI site. The
Pro allele is detected by an undigested PCR product of 500
bp, whereas the Arg allele produces two bands of 318 and 182
bp.
RSPs were determined in 73 unrelated healthy controls; the genotype
frequencies are summarized in Fig. 1B
. Genotype and allelic frequencies
for the Mediterranean population analyzed do not statistically differ
from frequencies reported in the ECTIM study (3)
(P values not shown for the sake of simplicity). The
frequency of the allele for the codon 10 polymorphism was 0.47% among
healthy Japanese controls, 0.53% in the ECTIM, and 0.59% in the
present studies; the differences are not statistically significant.
The major advantage of this method is its simplicity because only two PCR reactions are used to genotype four RSPs. This procedure replaces the three pairs of primers used by Cambien et al. (3) to amplify and determine the same polymorphisms (codons 10 and 25 are also amplified in the same PCR reaction) and the design and temperature optimization of eight ASOs. It also reduces the processing time per sample because the ASO method not only needs PCR amplification but also needs 6 h of hybridization, without taking into account the time for autoradiography. Digestion with restriction enzymes and polyacrylamide electrophoresis can be performed in 5 h. The present restriction enzyme method also avoids the use of radioactivity (32P-labeled oligonucleotides).
Recently, a codon 10 polymorphism has been characterized by allele-specific amplification (5). Allele-specific amplification is based on two primers, each differing from the other in the terminal 3' nucleotide, which are specific for each allelic variant. Taq polymerase will not extend if a mismatch is present at the 3' end of the primer-template duplex. This method involves a limited effort because only one PCR reaction is necessary for each allele-specific primer. Direct visualization on agarose gels is the only postamplification processing. However, primer design and PCR conditions are critical because under some conditions, a single 3'-mismatched base permits amplification (6). Purine-purine and pyrimidine-pyrimidine mismatches are more refractory to extension than purine-pyrimidine mismatches; in such cases, additional mismatches can be introduced to destabilize the primer-template duplex and to increase specificity. Magnesium, dNTPs, Taq polymerase or primer concentrations, and the precise temperature of annealing must be carefully controlled to maintain the specificity of the reaction (7).
Another three polymorphisms were described by Cambien et al.
(3) in the TGFB1 gene. A CA transversion was
detected at position -988; this could be considered as a variant
because it was present in only 2 of 1000 individuals. A "C"
insertion was also reported at position +72 in the 5'-untranslated
region. This polymorphism is in mutual association with a codon 25
polymorphism (as shown by the strong linkage disequilibrium coefficient
of -1.0 reported); therefore, we decided to exclude it and to analyze
codon 25 polymorphisms. The +72 polymorphism is more difficult to
detect than the CG transversion at codon 25, the latter being easily
detected by the loss of an FseI site. This allows us to use
only one pair of primers to amplify codons 25 and 10 in one PCR
reaction. Another variant was reported in codon 263. In this case, a
CT transition produces a Thr263Ile
substitution, the Thr allele being much more frequent, with only 6.7%
of the subjects analyzed being heterozygous for this polymorphism. To
our knowledge, there is no available enzyme capable of recognizing this
substitution; therefore, the use of allele-specific primers or the
design of a modified primer introducing an artificial restriction site
(8) could be considered.
The genotyping system described here is simple, reliable, and relatively inexpensive when a large sample set needs to be genotyped, and it could be performed in laboratories without facilities for radioactivity. In the future, clinical studies on the TGFB1 polymorphisms could confirm suspicions of the crucial role of TGF-ß1 and genetic predisposition to several fibrotic diseases.
Acknowledgments
This work was supported in part by the following grants: Grant Pensa-Esteve/1998 from the Catalonian Society of Nephrology and Grant FIS 99/0176 from the Fondo de Investigaciones Sanitarias. We thank Drs. I. Hutchinson and V. Pravica (School of Biological Sciences, University of Manchester, Manchester, UK) for generously providing the sequences for the F3-F4 primers.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Xaubet, A. Marin-Arguedas, S. Lario, J. Ancochea, F. Morell, J. Ruiz-Manzano, E. Rodriguez-Becerra, J. M. Rodriguez-Arias, P. Inigo, S. Sanz, et al. Transforming Growth Factor-{beta}1 Gene Polymorphisms Are Associated with Disease Progression in Idiopathic Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., August 15, 2003; 168(4): 431 - 435. [Abstract] [Full Text] [PDF]
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