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Articles |
1
Kuopio University Hospital, Department of Clinical Chemistry, FIN-70211 Kuopio, Finland.
2
Kuopio University, Department of Clinical Chemistry,
FIN-70211 Kuopio, Finland.
3
Turku University Central Hospital, Department of
Clinical Chemistry and Hematology, FIN-20521 Turku, Finland.
a Address correspondence to this author at: Turku University Central Hospital, Department of Clinical Chemistry and Hematology, PO Box 52, FIN-20521 Turku, Finland. Fax 358-2-2613924; e-mail ilkka.mononen{at}tyks.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods and Results: We describe two different molecular methods for the detection of the NPHS1 mutations: a PCR-restriction fragment length polymorphism (PCR-RFLP) and a dual-color oligonucleotide ligation assay (OLA). The dual-color OLA, which enables simultaneous detection of the NPHS1 FinMajor and FinMinor mutations, can be used for rapid analysis of large sets of samples. The analysis of 2004 Finnish blood samples revealed 34 carriers of the FinMajor mutation and 1 carrier of the FinMinor mutation, indicating a carrier frequency of 1:59 (95% confidence interval, 1:89–1:44) for the NPHS1 FinMajor mutation and 1:2004 (95% confidence interval, 0 to 1:677) for the NPHS1 FinMinor mutation, respectively.
Conclusion: PCR-RFLP and dual-color OLA are suitable for molecular diagnosis and carrier screening of the major mutations that cause NPHS1.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein in the maternal serum or amniotic fluid
(4)(5). Recently, two major mutations in the
NPHS1 gene, which encodes the membrane protein nephrin, have
been described in the Finnish NPHS1 patients (6). Based on
the analysis of the affected neonates, 78% of the NPHS1
alleles have been reported to carry a 2-bp deletion in exon 2
(FinMajor), and 16% of the alleles carry a
nonsense mutation in exon 26 (FinMinor)
(6). The high prevalence of these two mutations simplifies
the use of molecular techniques for the diagnosis of NPHS1 in the
Finnish population. These techniques can also be used for the screening
of asymptomatic carriers of this disorder, e.g., to find couples at
risk of having an affected child. We have developed two molecular methods for the detection of the NPHS1 FinMajor and FinMinor mutations. In restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified target DNA, the FinMinor mutation abolishes a restriction site of the AvaII enzyme, thus preventing the digestion of the PCR product with this enzyme. The detection of the FinMajor mutation is based on the PCR-mediated generation of an artificial TaqI restriction site in the DNA template containing the FinMajor mutation. For the simultaneous detection of the FinMajor and FinMinor mutations from a large number of samples, we utilized PCR followed by oligonucleotide ligation assay (PCR-OLA) (7)(8). Both of the mutation sites were amplified in a single PCR reaction. The reporter probes for the FinMajor and FinMinor mutation sites were labeled with different haptens, thus allowing simultaneous detection of two mutation sites in a single microplate well by ELISA. The applicability of the dual-color PCR-OLA for carrier screening of the FinMajor and the FinMinor mutations of NPHS1 was tested by analyzing 2004 samples from eastern Finland.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis and modification of oligonucleotides
The oligonucleotides for the PCR and ligation reactions were
synthesized using standard phosphoramidite chemistry. For PCR-RFLP, the
target DNA in exon 2 (FinMajor site) was
amplified using primers 5'-TTCTGGGCCCTGCCTGAAATC-3' and
5'-CATCTTTGGCCCATTGCAC-3', and the target DNA in exon 26
(FinMinor site) was amplified using primers
5'-GGGGCTTGCATAGGGTCACT-3' and 5'-AGTGTCCCGCTCTCCTGTCC-3'. For PCR-OLA,
the PCR primers described by Kestilä et al. (6) were
used. The nucleotide sequences of the allele-specific and reporter
oligonucleotides for the ligation reactions of the
FinMajor and FinMinor
mutations, and the G3315A polymorphism site are shown in Table 1
. For detection of the sequence at the
FinMinor site, a mixture of two reporter probes
(reporters 1 and 2) was used in the ligation reactions because of the
polymorphism in the region of the reporter probe. The allele-specific
oligonucleotides for OLA were modified with a 5'-biotin during the
synthesis. All reporter oligonucleotides were 5'-phosphorylated, and
the reporter probes for the FinMajor site and the
polymorphic site were 3'-digoxigenin labeled, whereas the reporter
probes for the FinMinor site were 3'-fluorescein
labeled by terminal transferase tailing reaction (8).
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pcr-rflp for detection of the FinMinor ANDFinMajor MUTATIONS
The PCR reaction mixtures contained 25 ng of genomic DNA or 0.5
µL of Split Second DNA sample, 50 ng of the amplification primers for
the FinMinor or the
FinMajor mutation site, 6 nmol of each four
deoxynucleotide triphosphates, 0.5 U of DyNAZyme II thermostable DNA
polymerase (Finnzymes), 10 mmol/L Tris-HCl (pH 8.8 at 25 °C), 1.5
mmol/L MgCl2, 50 mmol/L KCl, and 1 mL/L
Triton-X-100 in a reaction volume of 30 µL. The PCR amplifications
were performed in an MJ Research PTC-100 thermal cycler (MJ
Research). The cycling protocol included an initial temperature
cycle of 95 °C for 5 min, 85 °C for the time needed to add the
enzyme, 58 °C for 30 s, and 72 °C for 45 s, followed by
a cycle of 94 °C for 30 s, 58 °C for 30 s, and 72 °C
for 45 s. A total of 30 cycles were performed, with final
extension at 72 °C for 10 min.
For detection of the FinMajor mutation by RFLP, 20 µL of a restriction enzyme mixture containing 3 U of TaqI enzyme (Promega), 1x Buffer E (6 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 6 mmol/L MgCl2, and 1 mmol/L dithiothreitol), and 0.1 g/L acetylated bovine serum albumin (BSA) was added directly to the PCR reaction mixture containing the 103-bp PCR product, and the mixture was incubated for 1 h at 65 °C. For detection of FinMinor mutation, 20 µL of restriction enzyme mixture containing 3 U of AvaII enzyme (Promega), 1x Buffer C (10 mmol/L Tris-HCl, pH 7.9, 50 mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L dithiothreitol), and 0.1 g/L acetylated BSA was added to the PCR reaction mixture containing the 239-bp PCR product, and the mixture was incubated for 2 h at 37 °C. After the digestion, the analyses were completed by electrophoresis in a 2.5% agarose gel.
dual-color pcr-ola for detection of the
FinMinor and
FinMajor MUTATIONS
For PCR-OLA, the target DNA in exons 2 and 26 of the
NPHS1 gene was simultaneously amplified by PCR. The dual-PCR
reaction mixture was similar to the PCR mixture for the PCR-RFLP, but
contained the amplification primers for both the
FinMinor and the FinMajor
mutation sites and 1.0 mmol/L MgCl2 in a reaction
volume of 30 µL. The PCR program consisted of an initial temperature
cycle of 95 °C for 5 min, 85 °C for the time needed to add the
enzyme, 60 °C for 1 min, and 68 °C for 2 min, followed by 30
cycles of 94 °C for 1 min, 60 °C for 1 min, 68 °C for 2 min,
and a final extension of 68 °C for 8 min.
The PCR mixtures containing the 491-bp (exon 2) and 611-bp (exon 26) PCR products were diluted 1:10 with 1 mL/L Triton X-100. The ligation reactions contained 10 µL of the 1:10-diluted PCR product mixture, 0.15 pmol of each allele-specific and reporter probe, 1.2 U of thermostable ligase (New England Biolabs), and 1x NE Buffer [20 mmol/L Tris-HCl (pH 7.6 at 25 °C), 20 mmol/L potassium acetate, 10 mmol/L magnesium acetate, 10 mmol/L dithiothreitol, 1 mmol/L NAD+, and 1 mL/L Triton X-100] in a total volume of 30 µL. The reaction mixtures were subjected to 10 cycles of 97 °C for 30 s and 63 °C for 2 min, and stopped by the addition of 15 µL of 1 mL/L Triton X-100 containing 0.1 mol/L EDTA. All pipetting steps in the OLA and ELISA were performed using an 8-channel pipette (Biohit) or a Tecan Genesis RSP 150 automated pipetting device (Tecan).
For the assay readout, the reaction mixtures were transferred into the wells of a streptavidin-coated, BSA-blocked microtiter plate, and the biotin-labeled allele-specific oligonucleotides were allowed to bind to the streptavidin for 45 min at room temperature. The PCR products and unligated reporter oligonucleotides were removed from the wells by washing the plate first with 10 mmol/L NaOH containing 0.5 mL/L Tween 20 and then with a washing buffer containing 140 mmol/L NaCl, 0.5 mL/L Tween 20, 10 mmol/L Tris-HCl, pH 7.4. Anti-digoxigenin-alkaline phosphatase (Roche) and anti-fluorescein-horseradish peroxidase (Roche) diluted 1:2500 with the washing buffer containing 10 g/L BSA were added to the wells and incubated 2 h at room temperature. The plate was washed with the washing buffer, and the ELISA amplification system (Life Technologies) was used for detection of the FinMajor allele. The plate was measured with a Tecan Spectrafluor spectrophotometer (Tecan) at 510 nm. To detect the FinMinor allele, the plate was washed, the ABTS substrate (Zymed) was added, and the absorbance was measured at 405 nm. The absorbance of the blank well (no DNA in PCR) was subtracted from the absorbance values of the sample wells. The ratio of the absorbance of the wild-type (WT) allele-specific reaction to the mutant allele-specific reaction was calculated to determine the genotype. DNA samples from a patient homozygous for the FinMajor allele or the FinMinor allele were used as controls in every PCR-OLA series.
pcr-ola for detection of the g3315a polymorphism
The DNA region containing the G3315A polymorphism in exon 26 was
amplified by PCR using the same conditions as for the RFLP analysis of
the FinMinor mutation. The ligation reactions
containing the WT allele-specific or the polymorphism
allele-specific probe and the reporter probe were similar to those used
for the dual-OLA reactions. The ligation temperature was 58 °C.
After the cycles, ELISA was performed to detect the digoxigenin-labeled
reporter probe as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The PCR product of a NPHS1 patient homozygous for the
FinMajor mutation is digested into two fragments
of 20 and 83 bp because of the artificial restriction site of
TaqI enzyme created by the PCR primer (Fig. 1
, lane 1). In
the case of a WT genotype, the undigested 103-bp fragment is
present (Fig. 1
, lane 3) and the carrier of the
FinMajor mutation can be identified by the
presence of both the 83-and 103-bp fragments (Fig. 1
, lane 2). PCR
amplification of DNA from a NPHS1 patient homozygous for the
FinMinor mutation (Fig. 1
, lane 4) and a
WT control generated a 239-bp product, which appeared slightly
larger on electrophoresis. The undigested DNA fragment can be seen on
the gel because of an abolished restriction site of AvaII
(Fig. 1
, lane 4), whereas for a healthy control, the PCR
product is digested into two fragments of 188 and 51 bp (Fig. 1
, lane
6). Heterozygous carriers of the FinMinor
mutation are identified by the presence of all the three fragments
(239, 188, and 51 bp; Fig. 1
, lane 5).
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During the development of PCR-OLA for the
FinMinor mutation site, the nucleotide sequence
analysis revealed a silent mutation (G3315A) located 10 nucleotides
upstream from the FinMinor mutation in exon 26 of
NPHS1. The nucleotide sequences of the antisense strands of
the WT and the polymorphic allele are shown in Fig. 2
. The prevalence of the polymorphism in the Finnish population
was estimated by analyzing 256 blood samples. Seventy-four of 256
samples showed a WT genotype (95% confidence interval, 60–88 of 256),
70 of 256 showed a homozygous genotype for the polymorphism (95%
confidence interval, 56–84 of 256), and the remaining 112 of 256
samples (95% confidence interval, 96–128 of 256) showed a
heterozygous genotype. The prevalence of the WT allele (G3315) was 51%
(95% confidence interval, 46–55%), and the prevalence of the A3315
allele was 49% (95% confidence interval, 45–54%). The result was in
overall agreement with the Hardy-Weinberg equilibrium. Because the
polymorphism has such a high prevalence in the studied population, it
must be taken into consideration when performing PCR-OLA. Although the
polymorphism is situated in the middle of the reporter probe (Table 1
),
it disturbs the ligation of the oligonucleotides, producing a weak or
absent signal in ELISA. Therefore two different reporter probes, one
specific for the WT sequence (G3315) and the other specific for the
polymorphic sequence (A3315), were used in both ligation reactions for
the detection of the WT and FinMinor alleles in
exon 26.
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A schematic overview of the dual-color PCR-OLA for the detection of
NPHS1 is illustrated in Fig. 3
. The dual-PCR step is followed by two separate ligation
reactions (Fig. 3
, ligation A for the WT alleles and ligation B for the
mutant alleles). The covalent ligation of the oligonucleotides, which
occurs in the case of the perfect complementarity between the target
DNA and the oligonucleotides, is detected by the color development in
ELISA. Seven possible outcomes in dual-color PCR-OLA for the detection
of the NPHS1 FinMajor and
FinMinor mutations are shown in Fig. 3
. A WT
individual is positive for reaction A for both of the mutation sites
(Fig. 3
, outcome 1). A NPHS1 patient homozygous for the
FinMajor mutation is positive for reaction B in
exon 2 and for reaction A in exon 26 (Fig. 3
, outcome 2).
Correspondingly, a patient homozygous for the
FinMinor mutation is positive for reaction A for
exon 2 and for reaction B for exon 26 (Fig. 3
, outcome 3). A compound
heterozygote carrying both of these mutations would give a positive
reaction in each of the four reactions (Fig. 3
, outcome 4). A carrier
of either of the mutations shows a positive signal for reactions A and
B in the exon involved and a positive signal for reaction A for the
unaffected exon (Fig. 3
, outcomes 5 and 6). A negative control or a
sample with a failure in PCR is negative for all of the reactions (Fig. 3
, outcome 7). In theory, two additional outcomes of the dual-color OLA
are also possible: a patient homozygous for one mutation may be a
carrier of the other mutation.
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For the PCR-OLA numeric readout, the blank absorbance value was subtracted from the absorbance of the samples and the ratio of the absorbance for the WT reaction to that of the mutant reaction was calculated for both of the mutations separately in each sample. For samples homozygous for the WT allele, the absorbance ratio of the A and B reactions was >5, whereas samples homozygous for the mutant allele produced a ratio of the A and B reactions <0.2. A carrier of the mutation is detected by an absorbance ratio of the A and B reactions of >0.2 but <5 for the exon involved. For both the FinMajor mutation site in exon 2 and the FinMinor mutation site in exon 26, the absorbance ratio of the WT reaction to the mutant reaction (A/B) in samples from healthy individuals was significantly higher than the corresponding ratio in samples with the heterozygous genotype (P <0.001, Mann–Whitney U-test).
The 2.5th, 50th (median), and 97.5th percentile absorbance values of
the WT genotype and the carriers of the FinMajor
allele or the FinMinor allele along with the
absorbance ratios of the WT reaction (A) to the mutant reaction (B) are
shown in Table 2
. Among 2004 Finnish blood samples, we found 35 carriers of
NPHS1, of whom 34 individuals were carriers of the
FinMajor allele and 1 was a carrier of the
FinMinor allele. All of the samples showing the
carrier genotype and 5% of the samples of the WT genotype were also
analyzed using PCR-RFLP, and the results of these two separate assays
were in perfect concordance. The calculated carrier frequency of the
NPHS1 FinMajor allele in the Finnish population
was 1:59 (95% confidence interval, 1:89–1:44) and that of the
FinMinor allele was 1:2004 (95%
confidence interval, 0 to 1:677). The rates of homozygosity for the
NPHS1 FinMajor and FinMinor
mutations predicted from the carrier frequencies observed in the
present study were 1:13 900 (95% confidence interval,
1:31 700–1:7740) and 1:16 100 000 (95% confidence interval, 0 to
1:1 830 000), respectively, under the Hardy-Weinberg conditions.
Because the FinMajor and
FinMinor mutations account for 94% of all
mutations causing NPHS1 in the Finnish population, the predicted
combined carrier frequency of NPHS1 was 1:54 (95% confidence interval,
1:80–1:41) and the frequency of NPHS1 patients was 1:11 700 (95%
confidence interval, 1:25 600–1:6720) in the population.
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We also studied all of the carriers of the NPHS1 FinMajor mutation (n = 34), the single carrier of the NPHS1 FinMinor mutation, and two NPHS1 patients (one homozygous for the FinMajor mutation and the other homozygous for the FinMinor mutation) for the presence of the A3315 polymorphism. We found that 17 of 34 carriers of the FinMajor mutation were also carriers of the polymorphism and that 4 of 34 were homozygous for the polymorphism. Based on this result, the frequency of the polymorphism allele is lower among the carriers of the FinMajor mutation (37%; 95% confidence interval, 25–48%) than among the rest of the population. The carrier of the FinMinor mutation was also a carrier of the polymorphism, but neither of the NPHS1 patients carried the polymorphism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein concentration
in maternal serum or amniotic fluid. The two most common mutations that
cause NPHS1 cover >94% of the NPHS1 chromosomes in the
Finnish population (6), thus enabling the specific prenatal
or neonatal diagnosis of 88% of NPHS1 cases by detection of those two
mutations. Moreover, detection of the carriers of these mutations can
be used to identify couples at risk of having a child affected by
NPHS1. The FinMajor and
FinMinor mutations seem to be rare in other
populations because among 35 NPHS1 patients from North America, North
Africa, and Europe, only 2 Swedish and 4 Finnish patients had the
mutations (9). PCR-RFLP can be used for the detection of mutations that cause the elimination or creation of a restriction site (10). The detection of the FinMinor allele is achieved by detecting the elimination of the naturally occurring AvaII site, but the detection of the FinMajor allele requires creation of an artificial restriction site for TaqI enzyme by the addition of a mismatch to the PCR primer. PCR-RFLP is a highly specific method, but the lack of automation makes it inefficient for analyzing large sets of samples.
PCR-OLA assay has many advantages in the detection of characterized mutations in large-scale screening programs (8). The use of several haptens enables the detection of several mutations in a single well, further increasing the throughput of the assay (11). Dual-color PCR-OLA with ELISA-based detection has been used previously for the genotyping of diallelic sequence variations (11) and the detection of mutations in the human immunodeficiency virus (12). This methodology has now been applied to the detection of the FinMajor allele located in exon 2 and the FinMinor allele located approximately 20 kb downstream in exon 26 of the NPHS1 gene. For the simultaneous detection of these alleles, the optimization of the PCR-OLA was required. It was necessary to use 1.0 mmol/L MgCl2 in the PCR reaction mixture and to add the DNA polymerase after the first prolonged denaturation step to prevent nonspecific amplification. Moreover, performing the elongation step at 68 °C instead of 72 °C was found to improve amplification.
Despite successful amplification in PCR, a weak or absent signal was detected in some samples for the WT allele at the FinMinor site when primers based on the WT sequence at that region (6) were used. The reason for the failure in OLA was found to be a neutral polymorphism (G3315A) in the region of reporter probe. The polymorphism affects the third base of a codon and has no effect on the amino acid sequence. However, it precludes the ligation of the oligonucleotides in OLA. This polymorphism has a very high prevalence in the studied population (allele frequency of 49%; 95% confidence interval, 45–54%). To obtain a positive signal for all of the amplified samples, equal amounts of two different reporter probes, one specific for the WT sequence (G3315) and the other specific for the sequence containing the A3315 polymorphism, were used in both ligation reactions.
The analysis of the A3315 polymorphism allele among the carriers of the NPHS1 FinMajor mutation revealed that its frequency is lower among the carriers of the FinMajor mutation (37%; 95% confidence interval, 25%–48%) than among those negative for the mutation. DNA samples from NPHS1 patients homozygous for the FinMajor and the FinMinor mutations are needed to accurately determine the polymorphism frequency on mutant chromosomes.
The detected carrier frequency (1:59; 95% confidence interval,
1:89–1:44) for the NPHS1 FinMajor allele was
similar to the previously reported frequency (6). This is
higher than the carrier frequency of the major mutations that cause
aspartylglycosaminuria or infantile neuronal ceroid
lipofuscinosis in the Finnish population (1:94–1:83 and 1:95,
respectively) (13)(14)(15). The detected carrier frequency for
the FinMinor allele, 1:2004 (95% confidence
interval, 0 to 1:677), is lower than the frequency reported in a study
of 49 patients (6). This may be attributable to local
differences in the occurrence of the NPHS1 alleles in the
Finnish population. Our results show that the
FinMajor allele is the prominent NPHS1
allele in eastern Finland, where the samples for this study were
collected. The calculated frequency of NPHS1 carriers in the studied
population, assuming that 94% of the NPHS1 mutations are
FinMajor and FinMinor
mutations (6), would be 1:54 (95% confidence interval,
1:80–1:41), predicting an incidence of 1:11 600 (95% confidence
interval, 1:25 600–1:6720) for NPHS1. This incidence corresponds to
earlier studies among Finnish population (1)(2),
but for reasons as yet unknown, it is much lower than the incidence of
NPHS1 cases (1:2400) detected in eastern Finland by prenatal
-fetoprotein screening (4)(5). These data
give further evidence that there may be local differences in the
distribution of the NPHS1 alleles and that other mutations
may be present in the population of eastern Finland.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein. Lancet 1976;II:123–5..
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