- Email: [email protected]
Protocol proposal for Friedreich ataxia molecular diagnosis using fluorescent and triplet repeat primed polymerase chain reaction
Protocol proposal for Friedreich ataxia molecular diagnosis using fluorescent and triplet repeat primed polymerase chain reaction LAIA RODRI´GUEZ-REVENGA, IRENE MADRIGAL, DOLORES JIME´NEZ, MAR XUNCLA, and CE`LIA BADENAS MONTSERRAT MILA, BARCELONA, SPAIN
Friedreich ataxia (FRDA) is the most common hereditary ataxia that is caused mainly by an unstable GAA trinucleotide expansion in the first intron of the frataxin gene. Molecular tests for FRDA diagnosis and carrier detection include polymerase chain reaction (PCR) for the GAA expansion, triplet repeat primed PCR (TP-PCR), and/or Southern blotting. TP-PCR is a method developed to detect trinucleotide expansions successfully applied to FRDA diagnosis. In our laboratory, we have included a PCR for the GAA expansion using fluorescent primers polymerase chain reaction (F-PCR) to identify normal heterozygous and affected individuals unambiguously. The purpose of our study was to reanalyze 310 samples previously diagnosed in our laboratory and compare the results with those obtained by F-PCR and TP-PCR. Eight percent of the discrepancies between the carrier and the normal individuals were identified correctly by this protocol. No discrepancy was detected in the affected individuals. These techniques are effective, and compared with Southern blotting, they are less labor-intensive and suitable for automation. We suggest a new routine protocol for FRDA diagnosis that includes F-PCR and TP-PCR. (Translational Research 2010;156:309–314) Abbreviations: EMQN ¼ European Molecular Genetics Quality Network; FRDA ¼ Friedreich ataxia; F-PCR ¼ florescent polymerase chain reaction; FXN ¼ frataxin; PCR ¼ polymerase chain reaction; TP-PCR ¼ triple repeat primed polymerase chain reaction
riedreich ataxia (FRDA; OMIM#229300) is the most common autosomal recessive hereditary ataxia, with a prevalence of 1 in 30,000 individuals in Western European populations.1,2 FRDA is caused mainly by an unstable expansion of a GAA trinucleotide repeat in the first intron of the frataxin (FXN) gene that is located on chromosome 9q13-q21.1.3 The number of repeats in normal alleles ranges from 5 to 33 (between 34 and 65 are considered premutated alleles), whereas in FRDA patients, it varies from 66 to 1,700 repeats, resulting in a reduced expression of the FXN gene.3
F
Molecular testing techniques for FRDA GAA expansion detection include polymerase chain reaction (PCR), long-range PCR (LR-PCR), triplet repeat primed PCR (TP-PCR), and/or Southern blotting. Protocols for FRDA diagnosis vary between laboratories. Based on the 2008 European Molecular Genetics Quality Network (EMQN) report on FRDA diagnosis, protocols for GAA expansion detection involve TP-PCR in 37% of laboratories and only PCR in 48%, whereas Southern blotting still is used as the only GAA expansion detection technique in 15% of laboratories (Fig 1).
From the Biochemistry and Molecular Genetics Service. Hospital Clı´nic; Fundacio´ Clı´nic per a la Recerca Biome`dica; CIBER de Enfermedades Raras; Institut d’Investigacions Biome`diques August Pi i Sunyer, Barcelona, Spain.
Reprint requests: Ce`lia Badenas, Biochemistry and Molecular Genetics Service, Villarroel 170, Hospital Clı´nic 08036 Barcelona, Spain; e-mail: [email protected].
Supported by the CIBER de Enfermedades Raras.
Ó 2010 Mosby, Inc. All rights reserved.
Submitted for publication May 6, 2010; revision submitted July 27, 2010; accepted for publication August 3, 2010.
doi:10.1016/j.trsl.2010.08.001
1931-5244/$ - see front matter
309
310
Translational Research November 2010
et al Xuncla
Southern Blotting 15%
AT A GLANCE COMMENTARY M, et al. Xuncla
TP-PCR 37%
PCR 48%
Background
Molecular tests for Friedreich ataxia (FRDA) diagnosis include polymerase chain reaction (PCR) for the GAA expansion, triplet repeat primed PCR (TP-PCR), and/or Southern blotting. PCR affords a cheap, rapid, and often reliable result. However, in some cases, it can provide false results. On the other hand, Southern blotting is an expensive and labor-intensive technology. The development of a reliable and rapid technology has led to the use of TP-PCR for molecular diagnosis. Translational Significance
We propose the inclusion of a fluorescent PCR and TP-PCR protocol that can be included in routine FRDA molecular diagnosis.
PCR for GAA amplification followed by agarose gel electrophoresis for allele sizing may have some problems like faint bands resembling long-range GAA expansions and allele drop out. The faint bands could correspond to unspecific amplifications or to different types of heteroduplex-like molecules, and a report of FRDA carrier could be given to a normal individual.4 On the other hand, a carrier individual can be diagnosed as normal when PCR fails to amplify the expanded allele.5 Moreover, agarose gel cannot discriminate between normal homozygous and heterozygous samples with similar allele sizes. In 1996, Warner et al6 proposed a TP-PCR developed to detect expanded CTG repeats in myotonic dystrophy. The method consists of a PCR reaction using a fluorescentlabeled locus-specific primer flanking the repeat, which dictates the specificity, and 2 paired primers amplifying from multiple priming sites within the repeat. TP-PCR gives a characteristic ladder on the fluorescence trace that enables the rapid identification of large expansion repeats. Successful application of the technique to routine protocol for testing diseases caused by trinucleotide expansions, including FRDA, has been described.7 Ciotti et al5 validated TP-PCR as a screening method for FRDA diagnosis by testing samples previously genotyped by Southern blotting. The utility of the method in a clinical setting, although it cannot give a precise estimation of the expansion size, already has been reported.7,8 Nevertheless, variability among individuals is very high, and it is not possible to predict the clinical severity based only on the GAA mutation.9
Fig 1. Methodologies used by different laboratories for FRDA diagnosis (based on 2008 EMQN report). (Color version of figure is available online.)
Our laboratory has included a PCR for GAA triplet amplification using a fluorescently labeled primer PCR (FPCR), gel agarose electrophoresis, and allele sizing using a sequencer. With this approach, normal heterozygous and affected individuals are identified clearly and correctly, and no further analysis is needed, although confirmation of affected individuals by TP-PCR is advisable. However, the limitations of large allele amplification and unspecific bands described earlier are not avoided with this approach. These limitations are overcome with the inclusion of TP-PCR in the diagnosis protocol. Since 2008, a protocol including F-PCR and TP-PCR has been used in our laboratory. F-PCR is used as an initial screening method in which affected and heterozygous normal individuals are detected. TP-PCR is performed in those samples in which only 1 allele is identified as well as to confirm affected individuals. The purpose of our study was to test the reliability of this protocol for FRDA by reanalyzing 310 samples previously received in our center (analyzed by nonfluorescent PCR and Southern blotting). METHODS Subjects. Molecular diagnosis for FRDA had been performed previously on 310 samples referred to the Biochemistry and Molecular Genetics Department of the Hospital Clı´nic of Barcelona between 1993 and 2007. Genomic DNA was obtained from blood samples using standard methods. The nonfluorescent PCR protocol for FRDA diagnosis (including 2 PCR reactions named short and long) and Southern blotting was performed as described elsewhere.3 The current study was approved by the Ethics Committee of Hospital Clinic (Barcelona). In the present study, all individuals were reanalyzed by F-PCR and gel agarose electrophoresis. Samples from unaffected individuals (272) were run on a sequencer, and those with a homozygous compatible pattern were included in the TP-PCR study. Affected individuals also were confirmed by TP-PCR. F-PCR. Forward primer for the short-PCR was labeled fluorescently with FAM (6-carboxyfluorescein), and
Translational Research Volume 156, Number 5
PCR was performed as described. 3 PCR products were run on an agarose gel (for detection and allele sizing of the affected and some carrier individuals) and afterward analyzed with an ABI3100 genetic analyzer (Applied Biosystems, Foster City, Calif). GeneMapper v3.5 software (Applied Biosystems) was used to analyze the results (the identification of normal heterozygous individuals). TP-PCR. TP-PCR was performed using the primers described by Ciotti et al5 in the 206 samples in which fluorescent short-PCR yielded only 1 allele. TP-PCR conditions were 1.5 mL containing 150 ng to 200 ng of genomic DNA, 1.8 mmol/L of MgCl2, 150 mmol/L of dNTPs, 0.5 mmol/L of primer P1, 0.5 mmol/L of primer P3, 0.05 mmol/L of primer P4, and 1 unit of Taq polymerase in 13 specific buffer (Finnzymes, Espoo, Finland) in a final volume of 22.35 mL. The cycling parameters were an initial denaturation of 5 min at 94 C, 30 cycles consisting of 30 s at 94 C, 30 s at 60 C, 30 s at 72 C, and a final extension of 10 min at 72 C. Analysis of F-PCR products was performed in an ABI3100 genetic analyzer. The following control samples also were analyzed in each PCR: a normal individual for FRDA (either homozygous or heterozygous), an expansion carrier, and an affected individual. Discordant results between TP-PCR and a previous study were performed twice.
et al Xuncla
311
310 samples
F-PCR (agarose gel)
38 affected
272 non-affected
TP-PCR
F-PCR (sequencer)
66 normal heterozygous
206 apparently homozygous
TP-PCR
151 normal homozygous
55 carriers
Fig 2. Samples referred for FRDA diagnosis included in the study.
11 individuals initially were misclassified as carriers when they were normal individuals (4 heterozygous and 7 homozygous).
RESULTS
DISCUSSION
We have reanalyzed 310 samples received by our center for FRDA molecular diagnosis by F-PCR. Results from the present study are summarized in Fig 2. Gel agarose electrophoresis allowed the identification of 38 affected individuals that afterward were confirmed by TP-PCR. Sequencer analysis of F-PCR from the 272 unaffected individuals identified 66 normal heterozygous, making any further study unnecessary and yielding a normal result for the GAA expansion. The remaining 206 samples were included in the TP-PCR study; 151 were normal homozygous individuals, whereas 55 were carriers. TP-PCR gave a characteristic pattern, allowing us to discriminate among affected, carrier, and normal individuals for FRDA. In some normal samples, we even could distinguish heterozygote genotypes as the TPPCR showed a different profile. Typical results from F-PCR and TP-PCR from normal, carrier, and affected individuals are shown in Fig 3. The results obtained with F-PCR and TP-PCR yielded a 92% concordance (285/310) with the previous 2-PCR results (either affected, normal, or carrier). No affected individual had been characterized wrongly. A total of 25 samples (8%) in which the present study varied the status; 14 were considered incorrectly as normal homozygous individuals when they were carriers, and
Homozygous expansion of the GAA repeat tract is the cause of FRDA in 98% of cases. In the remaining cases, a point mutation in the FXN coding region is detected in heterozygous carriers of GAA expansion.3 Southern blotting analysis had been used widely for the detection and sizing of expanded FXN alleles. Nevertheless, it is a labor-intensive and time-consuming technique that is not suitable for routine diagnosis when few samples are studied. Today, it is still used by 15% of laboratories as the only method for detecting GAA expansions in the FXN gene according to the 2008 EMQN report. TPPCR was introduced for FRDA diagnosis in 2004, and it has proved to be a useful technique for both small and large numbers of samples.5,7,8 TP-PCR assay is an easy and sensitive method for expanded allele detection of rapid execution and requires reduced amounts of DNA. Our study combined the use of F-PCR and TP-PCR for FRDA diagnosis in 310 samples that had been studied previously by long and short PCR. F-PCR allowed the diagnosis of 21.3% samples as normal heterozygous individuals that otherwise would have required further studies. TP-PCR yielded unambiguous results in the remaining 244 samples (Fig 2). Eight percent of the discrepancies were detected between the 2-PCR protocol and present results. Misclassified individuals were
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et al Xuncla
Translational Research November 2010
Fig 3. Example of FRDA results obtained by F-PCR and TP-PCR. Results from normal homozygous, 2 normal heterozygous (with similar and different GAA repeat number), carrier, and affected individuals for the GAA expansion in the FXN gene are shown. A, Gel agarose electrophoresis image showing the F-PCR products. No differences between normal (both homozygous and heterozygous) and carrier individuals are detected in these samples. An affected individual can be identified. B, F-PCR electropherogram. Normal heterozygous individuals are identified, whereas normal homozygous and carrier individuals cannot be distinguished. No amplification is obtained in the affected individuals. C, TP-PCR electropherogram. Characteristic patterns for normal, carrier, and affected individuals are shown. (Color version of figure is available online.)
normal individuals that previously had been considered carriers because of unspecific amplification bands (7 homozygous individuals) or heteroduplex-like molecules on agarose gel electrophoresis (4 heterozygous individuals), and carrier individuals previously were considered normal (14 individuals) in which amplification of the expanded allele had failed.
The algorithm for FRDA diagnosis in our laboratory is described in Fig 4. The first step of this protocol is F-PCR, which allows the correct identification of normal heterozygous samples and affected individuals. To confirm affected individuals as well as to characterize apparently homozygous samples, TP-PCR is performed. This method determines the
Translational Research Volume 156, Number 5
et al Xuncla
313
FRDA sample
F-PCR
2 normal alleles
1 normal allele or no amplification
Normal heterozygous
TP-PCR
Normal homozygous
Carrier
2 expanded alleles
Affected
Affected
Clinical data
No amplification
New sample
Clinically affected
Clinically unaffected (family study)
FXN Sequencing
Carrier
Fig 4. FRDA diagnosis algorithm. The first step for FRDA diagnosis includes F-PCR to all samples. If 2 normal alleles are detected, then a normal heterozygous report is given. When 2 amplified alleles are detected, TP-PCR should be performed in this sample to confirm the affected status. When only 1 normal allele or no amplification is obtained, TP-PCR is performed for the identification of normal homozygous and carrier individuals. If no amplification is obtained, then a new sample is required. When obtaining a carrier result in a clinically affected individual, FXN sequencing should be performed.
sample status of normal homozygous, carrier, or affected. In clinically affected individuals in which only 1 expanded allele is detected, sequencing of the FXN gene will be performed. When no amplification is obtained by F-PCR and TP-PCR, a new sample should be requested. In conclusion, the use of F-PCR followed by TP-PCR in selected samples could be used in routine protocol for FRDA diagnosis. This protocol is rapid, accurate, and suitable to automation; it could be used to screen numerous samples and it is cost effective. CIBERer is an initiative of the ISCIII (Instituto de Salud Carlos III).
REFERENCES
1. Delatycki MB, Williamson R, Forrest SM. Friedreich ataxia: an overview. J Med Genet 2000;37:1–8. 2. Schulz JB, Boesch S, Burk K, et al. Diagnosis and treatment of Friedreich ataxia: a European perspective. Nat Rev Neurol 2009; 5:222–34. 3. Campuzano V, Montermini L, Molto MD, et al. Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996;271:1423–7. 4. Poirier J, Ohshima K, Pandolfo M. Heteroduplexes may confuse the interpretation of PCR-based molecular tests for the Friedreich ataxia GAA triplet repeat. Hum Mutat 1999;13:328–30. 5. Ciotti P, Di Maria E, Bellone E, Ajmar F, Mandich P. Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia. J Mol Diagn 2004;6:285–9.
314
et al Xuncla
6. Warner JP, Barron LH, Goudie D, et al. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 1996;33:1022–6. 7. Cagnoli C, Michielotto C, Matsuura T, et al. Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay. J Mol Diagn 2004;6:96–100.
Translational Research November 2010
8. Lamba LD, Ciotti P, Giribaldi G, et al. Friedreich’s ataxia: a new mutation in two compound heterozygous siblings with unusual clinical onset. Eur Neurol 2009;61:240–3. 9. Santos R, Lefevre S, Sliwa D, Seguin A, Camadro JM, Lesuisse E. Friedreich ataxia: molecular mechanisms, redox considerations, and therapeutic opportunities. Antioxid Redox Signal 2010;13:651–90.
Friedreich ataxia (FRDA) is the most common hereditary ataxia that is caused mainly by an unstable GAA trinucleotide expansion in the first intron of the frataxin gene. Molecular tests for FRDA diagnosis and carrier detection include polymerase chain reaction (PCR) for the GAA expansion, triplet repeat primed PCR (TP-PCR), and/or Southern blotting. TP-PCR is a method developed to detect trinucleotide expansions successfully applied to FRDA diagnosis. In our laboratory, we have included a PCR for the GAA expansion using fluorescent primers polymerase chain reaction (F-PCR) to identify normal heterozygous and affected individuals unambiguously. The purpose of our study was to reanalyze 310 samples previously diagnosed in our laboratory and compare the results with those obtained by F-PCR and TP-PCR. Eight percent of the discrepancies between the carrier and the normal individuals were identified correctly by this protocol. No discrepancy was detected in the affected individuals. These techniques are effective, and compared with Southern blotting, they are less labor-intensive and suitable for automation. We suggest a new routine protocol for FRDA diagnosis that includes F-PCR and TP-PCR. (Translational Research 2010;156:309–314) Abbreviations: EMQN ¼ European Molecular Genetics Quality Network; FRDA ¼ Friedreich ataxia; F-PCR ¼ florescent polymerase chain reaction; FXN ¼ frataxin; PCR ¼ polymerase chain reaction; TP-PCR ¼ triple repeat primed polymerase chain reaction
riedreich ataxia (FRDA; OMIM#229300) is the most common autosomal recessive hereditary ataxia, with a prevalence of 1 in 30,000 individuals in Western European populations.1,2 FRDA is caused mainly by an unstable expansion of a GAA trinucleotide repeat in the first intron of the frataxin (FXN) gene that is located on chromosome 9q13-q21.1.3 The number of repeats in normal alleles ranges from 5 to 33 (between 34 and 65 are considered premutated alleles), whereas in FRDA patients, it varies from 66 to 1,700 repeats, resulting in a reduced expression of the FXN gene.3
F
Molecular testing techniques for FRDA GAA expansion detection include polymerase chain reaction (PCR), long-range PCR (LR-PCR), triplet repeat primed PCR (TP-PCR), and/or Southern blotting. Protocols for FRDA diagnosis vary between laboratories. Based on the 2008 European Molecular Genetics Quality Network (EMQN) report on FRDA diagnosis, protocols for GAA expansion detection involve TP-PCR in 37% of laboratories and only PCR in 48%, whereas Southern blotting still is used as the only GAA expansion detection technique in 15% of laboratories (Fig 1).
From the Biochemistry and Molecular Genetics Service. Hospital Clı´nic; Fundacio´ Clı´nic per a la Recerca Biome`dica; CIBER de Enfermedades Raras; Institut d’Investigacions Biome`diques August Pi i Sunyer, Barcelona, Spain.
Reprint requests: Ce`lia Badenas, Biochemistry and Molecular Genetics Service, Villarroel 170, Hospital Clı´nic 08036 Barcelona, Spain; e-mail: [email protected].
Supported by the CIBER de Enfermedades Raras.
Ó 2010 Mosby, Inc. All rights reserved.
Submitted for publication May 6, 2010; revision submitted July 27, 2010; accepted for publication August 3, 2010.
doi:10.1016/j.trsl.2010.08.001
1931-5244/$ - see front matter
309
310
Translational Research November 2010
et al Xuncla
Southern Blotting 15%
AT A GLANCE COMMENTARY M, et al. Xuncla
TP-PCR 37%
PCR 48%
Background
Molecular tests for Friedreich ataxia (FRDA) diagnosis include polymerase chain reaction (PCR) for the GAA expansion, triplet repeat primed PCR (TP-PCR), and/or Southern blotting. PCR affords a cheap, rapid, and often reliable result. However, in some cases, it can provide false results. On the other hand, Southern blotting is an expensive and labor-intensive technology. The development of a reliable and rapid technology has led to the use of TP-PCR for molecular diagnosis. Translational Significance
We propose the inclusion of a fluorescent PCR and TP-PCR protocol that can be included in routine FRDA molecular diagnosis.
PCR for GAA amplification followed by agarose gel electrophoresis for allele sizing may have some problems like faint bands resembling long-range GAA expansions and allele drop out. The faint bands could correspond to unspecific amplifications or to different types of heteroduplex-like molecules, and a report of FRDA carrier could be given to a normal individual.4 On the other hand, a carrier individual can be diagnosed as normal when PCR fails to amplify the expanded allele.5 Moreover, agarose gel cannot discriminate between normal homozygous and heterozygous samples with similar allele sizes. In 1996, Warner et al6 proposed a TP-PCR developed to detect expanded CTG repeats in myotonic dystrophy. The method consists of a PCR reaction using a fluorescentlabeled locus-specific primer flanking the repeat, which dictates the specificity, and 2 paired primers amplifying from multiple priming sites within the repeat. TP-PCR gives a characteristic ladder on the fluorescence trace that enables the rapid identification of large expansion repeats. Successful application of the technique to routine protocol for testing diseases caused by trinucleotide expansions, including FRDA, has been described.7 Ciotti et al5 validated TP-PCR as a screening method for FRDA diagnosis by testing samples previously genotyped by Southern blotting. The utility of the method in a clinical setting, although it cannot give a precise estimation of the expansion size, already has been reported.7,8 Nevertheless, variability among individuals is very high, and it is not possible to predict the clinical severity based only on the GAA mutation.9
Fig 1. Methodologies used by different laboratories for FRDA diagnosis (based on 2008 EMQN report). (Color version of figure is available online.)
Our laboratory has included a PCR for GAA triplet amplification using a fluorescently labeled primer PCR (FPCR), gel agarose electrophoresis, and allele sizing using a sequencer. With this approach, normal heterozygous and affected individuals are identified clearly and correctly, and no further analysis is needed, although confirmation of affected individuals by TP-PCR is advisable. However, the limitations of large allele amplification and unspecific bands described earlier are not avoided with this approach. These limitations are overcome with the inclusion of TP-PCR in the diagnosis protocol. Since 2008, a protocol including F-PCR and TP-PCR has been used in our laboratory. F-PCR is used as an initial screening method in which affected and heterozygous normal individuals are detected. TP-PCR is performed in those samples in which only 1 allele is identified as well as to confirm affected individuals. The purpose of our study was to test the reliability of this protocol for FRDA by reanalyzing 310 samples previously received in our center (analyzed by nonfluorescent PCR and Southern blotting). METHODS Subjects. Molecular diagnosis for FRDA had been performed previously on 310 samples referred to the Biochemistry and Molecular Genetics Department of the Hospital Clı´nic of Barcelona between 1993 and 2007. Genomic DNA was obtained from blood samples using standard methods. The nonfluorescent PCR protocol for FRDA diagnosis (including 2 PCR reactions named short and long) and Southern blotting was performed as described elsewhere.3 The current study was approved by the Ethics Committee of Hospital Clinic (Barcelona). In the present study, all individuals were reanalyzed by F-PCR and gel agarose electrophoresis. Samples from unaffected individuals (272) were run on a sequencer, and those with a homozygous compatible pattern were included in the TP-PCR study. Affected individuals also were confirmed by TP-PCR. F-PCR. Forward primer for the short-PCR was labeled fluorescently with FAM (6-carboxyfluorescein), and
Translational Research Volume 156, Number 5
PCR was performed as described. 3 PCR products were run on an agarose gel (for detection and allele sizing of the affected and some carrier individuals) and afterward analyzed with an ABI3100 genetic analyzer (Applied Biosystems, Foster City, Calif). GeneMapper v3.5 software (Applied Biosystems) was used to analyze the results (the identification of normal heterozygous individuals). TP-PCR. TP-PCR was performed using the primers described by Ciotti et al5 in the 206 samples in which fluorescent short-PCR yielded only 1 allele. TP-PCR conditions were 1.5 mL containing 150 ng to 200 ng of genomic DNA, 1.8 mmol/L of MgCl2, 150 mmol/L of dNTPs, 0.5 mmol/L of primer P1, 0.5 mmol/L of primer P3, 0.05 mmol/L of primer P4, and 1 unit of Taq polymerase in 13 specific buffer (Finnzymes, Espoo, Finland) in a final volume of 22.35 mL. The cycling parameters were an initial denaturation of 5 min at 94 C, 30 cycles consisting of 30 s at 94 C, 30 s at 60 C, 30 s at 72 C, and a final extension of 10 min at 72 C. Analysis of F-PCR products was performed in an ABI3100 genetic analyzer. The following control samples also were analyzed in each PCR: a normal individual for FRDA (either homozygous or heterozygous), an expansion carrier, and an affected individual. Discordant results between TP-PCR and a previous study were performed twice.
et al Xuncla
311
310 samples
F-PCR (agarose gel)
38 affected
272 non-affected
TP-PCR
F-PCR (sequencer)
66 normal heterozygous
206 apparently homozygous
TP-PCR
151 normal homozygous
55 carriers
Fig 2. Samples referred for FRDA diagnosis included in the study.
11 individuals initially were misclassified as carriers when they were normal individuals (4 heterozygous and 7 homozygous).
RESULTS
DISCUSSION
We have reanalyzed 310 samples received by our center for FRDA molecular diagnosis by F-PCR. Results from the present study are summarized in Fig 2. Gel agarose electrophoresis allowed the identification of 38 affected individuals that afterward were confirmed by TP-PCR. Sequencer analysis of F-PCR from the 272 unaffected individuals identified 66 normal heterozygous, making any further study unnecessary and yielding a normal result for the GAA expansion. The remaining 206 samples were included in the TP-PCR study; 151 were normal homozygous individuals, whereas 55 were carriers. TP-PCR gave a characteristic pattern, allowing us to discriminate among affected, carrier, and normal individuals for FRDA. In some normal samples, we even could distinguish heterozygote genotypes as the TPPCR showed a different profile. Typical results from F-PCR and TP-PCR from normal, carrier, and affected individuals are shown in Fig 3. The results obtained with F-PCR and TP-PCR yielded a 92% concordance (285/310) with the previous 2-PCR results (either affected, normal, or carrier). No affected individual had been characterized wrongly. A total of 25 samples (8%) in which the present study varied the status; 14 were considered incorrectly as normal homozygous individuals when they were carriers, and
Homozygous expansion of the GAA repeat tract is the cause of FRDA in 98% of cases. In the remaining cases, a point mutation in the FXN coding region is detected in heterozygous carriers of GAA expansion.3 Southern blotting analysis had been used widely for the detection and sizing of expanded FXN alleles. Nevertheless, it is a labor-intensive and time-consuming technique that is not suitable for routine diagnosis when few samples are studied. Today, it is still used by 15% of laboratories as the only method for detecting GAA expansions in the FXN gene according to the 2008 EMQN report. TPPCR was introduced for FRDA diagnosis in 2004, and it has proved to be a useful technique for both small and large numbers of samples.5,7,8 TP-PCR assay is an easy and sensitive method for expanded allele detection of rapid execution and requires reduced amounts of DNA. Our study combined the use of F-PCR and TP-PCR for FRDA diagnosis in 310 samples that had been studied previously by long and short PCR. F-PCR allowed the diagnosis of 21.3% samples as normal heterozygous individuals that otherwise would have required further studies. TP-PCR yielded unambiguous results in the remaining 244 samples (Fig 2). Eight percent of the discrepancies were detected between the 2-PCR protocol and present results. Misclassified individuals were
312
et al Xuncla
Translational Research November 2010
Fig 3. Example of FRDA results obtained by F-PCR and TP-PCR. Results from normal homozygous, 2 normal heterozygous (with similar and different GAA repeat number), carrier, and affected individuals for the GAA expansion in the FXN gene are shown. A, Gel agarose electrophoresis image showing the F-PCR products. No differences between normal (both homozygous and heterozygous) and carrier individuals are detected in these samples. An affected individual can be identified. B, F-PCR electropherogram. Normal heterozygous individuals are identified, whereas normal homozygous and carrier individuals cannot be distinguished. No amplification is obtained in the affected individuals. C, TP-PCR electropherogram. Characteristic patterns for normal, carrier, and affected individuals are shown. (Color version of figure is available online.)
normal individuals that previously had been considered carriers because of unspecific amplification bands (7 homozygous individuals) or heteroduplex-like molecules on agarose gel electrophoresis (4 heterozygous individuals), and carrier individuals previously were considered normal (14 individuals) in which amplification of the expanded allele had failed.
The algorithm for FRDA diagnosis in our laboratory is described in Fig 4. The first step of this protocol is F-PCR, which allows the correct identification of normal heterozygous samples and affected individuals. To confirm affected individuals as well as to characterize apparently homozygous samples, TP-PCR is performed. This method determines the
Translational Research Volume 156, Number 5
et al Xuncla
313
FRDA sample
F-PCR
2 normal alleles
1 normal allele or no amplification
Normal heterozygous
TP-PCR
Normal homozygous
Carrier
2 expanded alleles
Affected
Affected
Clinical data
No amplification
New sample
Clinically affected
Clinically unaffected (family study)
FXN Sequencing
Carrier
Fig 4. FRDA diagnosis algorithm. The first step for FRDA diagnosis includes F-PCR to all samples. If 2 normal alleles are detected, then a normal heterozygous report is given. When 2 amplified alleles are detected, TP-PCR should be performed in this sample to confirm the affected status. When only 1 normal allele or no amplification is obtained, TP-PCR is performed for the identification of normal homozygous and carrier individuals. If no amplification is obtained, then a new sample is required. When obtaining a carrier result in a clinically affected individual, FXN sequencing should be performed.
sample status of normal homozygous, carrier, or affected. In clinically affected individuals in which only 1 expanded allele is detected, sequencing of the FXN gene will be performed. When no amplification is obtained by F-PCR and TP-PCR, a new sample should be requested. In conclusion, the use of F-PCR followed by TP-PCR in selected samples could be used in routine protocol for FRDA diagnosis. This protocol is rapid, accurate, and suitable to automation; it could be used to screen numerous samples and it is cost effective. CIBERer is an initiative of the ISCIII (Instituto de Salud Carlos III).
REFERENCES
1. Delatycki MB, Williamson R, Forrest SM. Friedreich ataxia: an overview. J Med Genet 2000;37:1–8. 2. Schulz JB, Boesch S, Burk K, et al. Diagnosis and treatment of Friedreich ataxia: a European perspective. Nat Rev Neurol 2009; 5:222–34. 3. Campuzano V, Montermini L, Molto MD, et al. Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996;271:1423–7. 4. Poirier J, Ohshima K, Pandolfo M. Heteroduplexes may confuse the interpretation of PCR-based molecular tests for the Friedreich ataxia GAA triplet repeat. Hum Mutat 1999;13:328–30. 5. Ciotti P, Di Maria E, Bellone E, Ajmar F, Mandich P. Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia. J Mol Diagn 2004;6:285–9.
314
et al Xuncla
6. Warner JP, Barron LH, Goudie D, et al. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 1996;33:1022–6. 7. Cagnoli C, Michielotto C, Matsuura T, et al. Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay. J Mol Diagn 2004;6:96–100.
Translational Research November 2010
8. Lamba LD, Ciotti P, Giribaldi G, et al. Friedreich’s ataxia: a new mutation in two compound heterozygous siblings with unusual clinical onset. Eur Neurol 2009;61:240–3. 9. Santos R, Lefevre S, Sliwa D, Seguin A, Camadro JM, Lesuisse E. Friedreich ataxia: molecular mechanisms, redox considerations, and therapeutic opportunities. Antioxid Redox Signal 2010;13:651–90.