Validation of a Commercially Available Screening Tool for the Rapid ...

11 downloads 1780 Views 1MB Size Report
Recently developed PCR-based methods for fragile X syndrome testing are ... The clinical performance of the assay was established with 528 blinded and previously analyzed ...... Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A,.
The Journal of Molecular Diagnostics, Vol. 17, No. 3, May 2015

jmd.amjpathol.org

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1 Grace X.Y. Lim,* Yu Ling Loo,* Farmaditya E.P. Mundhofir,y Ferdy K. Cayami,y Sultana M.H. Faradz,y Indhu-Shree Rajan-Babu,z Samuel S. Chong,zx Yvonne Y. Koh,* and Ming Guan* From the Biofactory Pte Ltd.,* Singapore, Republic of Singapore; the Division of Human Genetics,y Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Semarang, Indonesia; the Department of Pediatrics,z Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore; and the Khoo Teck Puat - National University Children’s Medical Institute,x National University Health System, Singapore, Republic of Singapore Accepted for publication December 22, 2014. Address correspondence to Yvonne Y. Koh, Ph.D., The Biofactory Pte Ltd., Blk 79, Ayer Rajah Crescent, #05-06/ 06, Singapore 139955. E-mail: [email protected].

Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%e100%) and 99.6% specificity (95% CI, 98.5%e99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates postPCR handling for all non-expanded samples, and exemplifies a truly efficient screening procedure. (J Mol Diagn 2015, 17: 302e314; http://dx.doi.org/10.1016/j.jmoldx.2014.12.005)

Fragile X syndrome (FXS) is the most common cause of inheritable intellectual disabilities and autism, affecting an estimated 1 in 4000 males and 1 in 5000 to 8000 females.1,2 The medical condition is attributed to a trinucleotide CGG repeat expansion (>200 CGG repeats, full mutation) in the 50 untranslated region of the fragile X mental retardation 1 (FMR1) gene, which results in aberrant hypermethylation of the FMR1 promoter.3e5 The CGG repeat expansion varies considerably in size among persons, giving rise to a spectrum of clinical outcomes through different pathogenic mechanisms.6e9 According to the American College of Medical Genetics, there are four FMR1 allelic forms: normal (NL), with 5 to 44 repeats; gray zone (GZ), with 45 to 54 repeats; premutation (PM), with 55 to 200 repeats; and full mutation (FM), with >200 repeats.10 Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.12.005

Persons with a FM allele of >200 CGG repeats usually have intellectual disabilities and present with symptoms of autism spectrum disorder.8,11 Other phenotypic presentations pertain to behavior, cognition, hyperactivity, language deficits, aggressions, and psychiatric problems such as social anxiety and withdrawal. Physical traits such as an elongated face, prominent ears, flat feet, and macroorchidism in puberty are also apparent.2,8,12 Disclosures: G.X.Y.L., Y.L.L., Y.Y.K., and M.G. are employees of The Biofactory Pte Ltd. The Biofactory supplied the commercial kits and sponsored equipment-related laboratory charges specific for this study. S.S.C. is one of the inventors of the technology that combined TP-PCR and melt curve analysis for rapid detection of trinucleotide repeat expansions (patent application number PCT/SG2010/000396). The remaining authors declared no disclosures.

Screening CGG Repeat Expansions in FMR1 Male and female persons with FMR1 PM alleles (55 to 200 CGG repeats) are carriers because they do not present symptoms of FXS. However, PM alleles are meiotically unstable and can expand to FM within one generation through females.13,14 Initial concerns for carriers, especially females, were focused on the intergenerational instability of expanded alleles rather than clinical presentations.11 Accumulated evidence later found that PM carriers may develop fragile X-associated conditions such as fragile Xeassociated tremor/ataxia syndrome9 and fragile Xeassociated primary ovarian insufficiency, a condition defined as menopause before 40 years of age.15 Female PM carriers have an estimated 20% risk of fragile Xeassociated primary ovarian insufficiency,12 whereas approximately 8% of female PM carriers and 40% of male PM carriers develop fragile Xeassociated tremor/ataxia syndrome.16 Some studies now suggest that PM carriers may have various degrees of learning disabilities and anxiety disorders.17e19 The consequence of carrying the FMR1 GZ allele is less apparent than with PM and FM alleles.2 Recent findings suggest that persons with FMR1 GZ alleles may have increased risks of developing adult-onset fragile X-associated conditions.20 However, more extensive studies are necessary to establish the effects of GZ alleles. Early diagnosis of FXS at a young age enables timely therapeutic interventions that can considerably improve quality of life.2,21 Because of the defined phenotypic presentations associated with various FMR1 allelic forms, molecular diagnoses of FXS and associated disorders are enabled by detecting CGG expansions in FMR1.21,22 Existing approaches for detecting such aberrant expansions include Southern blot analysis and various permutations of PCR tests.6,10,22,23 Southern blot analysis is notoriously labor-intensive, timeconsuming, and requires large amounts of DNA. The workflow is not optimized for high-throughput testing because limited numbers of samples can be processed simultaneously.6,24 As a result, various PCR-based methods were developed over the past decade as alternatives to Southern blot analysis.25,26 PCR approaches designed to probe for CGG repeat expansions in FMR1, regardless of permutation, are typically either conventional PCR or triplet repeat-primed PCR (TP-PCR). Conventional PCR uses primers that target the flanking sequence of the CGG repeat region.22,24,27 TP-PCR includes flanking and additional primers that target within the trinucleotide repeat region.6,22,28 Such PCR approaches have advantages and inherent limitations compared with Southern blot analysis. For instance, conventional PCR is ineffective in amplifying large CGG repeats because of the repetitive nature of the region and the high GC-rich content. The reaction often fails beyond a certain size range, resulting in nonamplification of large PM or FM alleles.22,24,27 In fact, such a shortcoming known as the null-allele was proposed as a rapid negative identification of CGG expansions in male samples.25 Because of the nonamplification of larger alleles in conventional PCR, heterozygous female specimens with one NL allele and a

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

Table 1 Genotypes of DNA Samples from Coriell Cell Repositories Used in Blinded Study (n Z 35) CGG repeats reported by Coriell, n Coriell sample ID Normal alleles (30 CGG repeats) GM06895 GM07538 GM06904 GM07543 GM06890* GM06911 GM06889 High normal alleles (30e44 CGG repeats) GM20244* GM20243 Gray zone alleles(45e54 CGG repeats) GM20232 GM20234 GM20230* GM20236 Premutation/full mutation alleles (55 CGG repeats) CD00014 GM06905 GM20242 GM06910 GM20231 GM06894 GM20240 GM06892 GM06907 GM06906 GM20241 GM06903 GM06896 GM06968 GM20237 GM06891 GM20233 GM20239 GM06852 GM07537 GM06897 GM07862 GM04025 GM05847 GM09237

Sex

Allele 1

M F F F M F F

23 29 29 29 30 30 30

M F

41 41

M F M F

46 46 53 53

M F F F M F F M F M F F F F M M M F M F M M M F M

56 70 73 75e89 76 78 80 80e85 85 85e90 93e110 95 95e140 107 100e104 100e117 117 183e193 >200 >200 477 501e550 645 w650 931e940

Allele 2

29 23 20 NL 23

29

31 31

23 30 30 30 30 29 29 23 23 23

20 28e29

21

*Samples are used as cutoff reference controls. F, female; M, male; NL, normal.

second expanded allele that does not amplify (one normal band after PCR) cannot be distinguished from homozygous female samples with two NL alleles (one normal band).23 Additional testing is unavoidable in such situations; exclusion of the presence of an expanded allele is necessary

303

Lim et al because approximately 40% females are homozygous NL.23,25 TP-PCR overcomes the inadequacies of conventional PCR by priming from within the CGG repeat region and enabling amplification of large PM or FM alleles. Although full-length amplicons of large expansions may not be obtained, amplicons up to a certain size range will still be generated and subsequently analyzed. Regardless of approach, both conventional PCR and TP-PCR require coupling with either gel or capillary electrophoresis to resolve amplicons of varying sizes.23,25 In theory, existing PCR approaches are considered screening tools that reduce reliance on Southern blot analysis.6,23,25 In practice, however, a true screening tool with a PCR-based workflow should ideally be streamlined and not require additional post-PCR processing or expensive equipment. Here, we validate the FastFraX FMR1 Identification kit (the FastFraX ID kit) as a screening tool for the rapid detection of CGG expansions in the FMR1 gene. The underlying technology was developed on the basis of a previous study by Teo et al.29 The assay uses a direct TP-PCR, coupled with melting curve analysis (MCA) for detecting CGG expansions. The melting temperatures of the PCR amplicons are determined from the melt curve profile and depend on amplicon size. Through a simple MCA and without post-PCR processing, samples are rapidly classified as having non-expanded (55 CGG repeats) alleles. Our study aims to verify the accuracy and effectiveness of the TP-PCR with MCA approach as a first-line PCR-only screening tool in detecting CGG repeat expansions in clinical samples.

Figure 1 Frequency of CGG repeat lengths in the clinical archive cohort of 528 samples. In samples with two alleles such as female heterozygous specimens or male mosaic samples, only the larger allele is considered in the analysis. FMR1, fragile X mental retardation 1 gene.

Materials and Methods

was mixed with a male PM sample (GM06906; 85 to 90 CGG repeats) or a FM sample (GM06852; >200 CGG repeats) to create simulated mosaic content of different concentrations. These samples were mixed in various proportions, yet maintained at a total DNA input of 50 ng per reaction. The resulting simulated mosaic samples contained 1%, 2.5%, 5%, 7.5%, 10%, 20%, 50%, and 100% of the PM or FM sample. Melt curve profiles of the simulated mosaic samples were compared with that of 53 CGG repeat control (GM20230) and 30 CGG repeat control (GM06890).

DNA Samples

DNA Samples from Clinical Archive

Thirty-eight individual cell line-derived DNA reference samples with various CGG repeat lengths were acquired from the Coriell Cell Repositories (Camden, NJ) (Table 1). Of these, three male DNA samples (GM06890, GM20244, and GM20230) with 30, 41, and 53 FMR1 CGG repeats were used for the initial assessment as recommended controls for the TP-PCR assay with MCA. The study included testing the analytic sensitivity, analytic specificity, and consistency of the assay when used on different PCR assay platforms. The genotypes of all Coriell DNA samples tested in this study are listed in Table 1. All 38 samples (Table 1) were used in an initial blinded (G.X.Y.L.) small-scale study to evaluate performance of the TP-PCR assay. The analytic specificity of the TP-PCR assay was evaluated with a DNA sample (GM23378 from Coriell Cell Repositories) of no direct relevance to FXS to test for potential interference. GM23378 (Coriell Cell Repositories) harbors a CTG repeat expansion in the dystrophia myotonica-protein kinase gene. To evaluate the sensitivity of the TP-PCR assay to detect mosaicism, a male NL sample (GM06890; 30 CGG repeats)

The performance of the TP-PCR assay with MCA was ultimately validated in a blinded (G.X.Y.L. and Y.L.L.) study of 528 archived clinical samples from an Indonesian population with intellectual disabilities. These genomic DNA samples were originally isolated from whole blood by using salting out methods as described,30 with slight modifications. The samples were previously characterized for FMR1 CGG repeat length by using a combination of the following methods: a two-primer conventional (flanking) PCR,5 followed by fragment length analysis on an ABI Prism 3730 DNA Analyzer (Life Technologies, Carlsbad, CA) with the GeneMapper version 4.0 (Apache, Los Angeles, CA) or Southern blot analysis.31 For samples with NL or PM alleles, exact CGG repeat lengths were determined with a combination of PCR and fragment analysis. For samples with results indicative of a FM, or for female samples that produced a single PCR product, Southern blot analysis was performed to confirm the FM or to differentiate between female heterozygous and homozygous samples.31 In a few cases, the confirmation was based on TP-PCR as

304

jmd.amjpathol.org

-

The Journal of Molecular Diagnostics

Screening CGG Repeat Expansions in FMR1

Figure 2

Melt curve profiles that show analytic sensitivity of the triplet repeat-primed PCR assay with melting curve analysis on the ABI 7500 Fast real-time PCR platform. Melt curve profiles of male (A) and female (B) Coriell DNA samples that cover four FMR1 allelic forms tested in varying amounts, from 10 ng to 100 ng per reaction. Each reaction was conducted in triplicates; representative profiles are shown. The resumed baseline dF/dT temperature cutoffs refer to the GZ samples for each sex: GM20230 (A) and GM20236 (B). FM, full mutation; FMR1, fragile X mental retardation 1 gene; GZ, gray zone; NL, normal; NTC, no-template control; PM, premutation; resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed.

previously described.25 The frequency of CGG repeat lengths in the archived cohort was tabulated on the basis of the collective results generated by the aforementioned approaches (Figure 1). Genomic DNA concentrations were requantified with a NanoVue Plus spectrophotometer (GE Healthcare, Little Chalfont, UK). Usage of these archived clinical samples for the present study was approved by the Medical Ethical Committee of the Faculty of Medicine (Diponegoro University, Semarang, Indonesia). All samples were stored at 20 C until use.

TP-PCR Assay with MCA The FastFraX ID kits (labeled For Research Use) were obtained from the Biofactory Pte Ltd (Singapore, Republic of Singapore). The kit was developed on the basis of a previous study.29 It uses a direct TP-PCR, which includes a combination of primers that target a flanking region and from within the CGG repeats. The primer mix was designed to amplify from the 30 end of the FMR1 CGG repeat region of non-modified genomic DNA.29 The required PCR mixture and DNA polymerase were also included in the kit. TP-PCR assays were performed according to the manufacturer’s instructions in 25-mL volumes with 50 ng of genomic DNA per test. The PCR assays that assessed

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

analytic performance were conducted in triplicates. Unless otherwise specified, PCR assays and subsequent MCAs were performed with the Rotor-Gene Q HRM (Qiagen, Hilden, Germany) at the Center for Biomedical Research of Diponegoro University. The thermal cycling conditions comprised an initial denaturation step at 95 C for 15 minutes, followed by 40 cycles of 99 C for 2 minutes, 65 C for 2 minutes, 72 C for 3 minutes, and then a final extension step at 72 C for 10 minutes. To study the potential variations of different PCR systems, a few sets of assays were also performed under identical thermal cycling conditions on the LightCycler 480 (Roche Diagnostics, Mannheim, Germany) or ABI 7500 Fast Real-Time PCR system (Life Technologies). PCR amplicons were automatically melted after thermal cycling for MCA. The MCA parameters on the Rotor-Gene Q HRM comprised a denaturation step at 95 C for 1 minute and a temperature ramp from 75 C to 99 C at a rate of 0.5 C with a 5-second hold at each step. The MCA parameters on the LightCycler 480 comprised a denaturation step at 95 C for 1 minute and a temperature ramp from 60 C to 99 C at a rate of 0.01 C/second with 50 acquisitions per  C. The MCA parameters on the ABI 7500 Fast comprised a denaturation step at 95 C for 1 minute and a temperature ramp from 60 C to 99.9 C at a ramp percentage of 0.5%.

305

Lim et al the generated melt curve profiles were visually checked for aberrant results (eg, significantly lower signals than the controls). The temperatures at which baseline negative first derivative of fluorescence versus temperature resumed (resumed baseline dF/dT temperature) were extracted from the melt curve profiles of cutoff controls. Samples were classified as nonexpanded, expanded, or indeterminate on the basis of their respective resumed baseline dF/dT temperatures relative to that of the cutoff control used. The classification data for the clinical archived samples were sorted with Microsoft Excel 2010 (Microsoft Corp, Redman, WA). Indeterminate and expanded samples warrant further testing and are considered positive detections by the TP-PCR assay.

Results Analytic Sensitivity

Figure 3

Melt curve profiles that show the analytic specificity of the triplet repeat-primed PCR assay with melting curve analysis on the ABI 7500 Fast real-time PCR platform. Melt curve profiles of four female DNA samples from Coriell spiked with increasing amounts of non-relevant DNA (GM23378) that harbor expanded CTG repeats in the DMPK gene. Zero, 50, 100, or 150 ng of GM23378 was added to 50 ng of each female DNA sample in each reaction. Each reaction was conducted in triplicates; representative profiles are shown. The resumed baseline dF/dT temperature cutoff refers to the GZ sample, GM20236. DMPK, dystrophia myotonica-protein kinase gene; FM, full mutation; GZ, gray zone; NL, normal; NTC, no-template control; PM, premutation; resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed.

Four Coriell DNA samples representing the male NL, GZ, PM, and FM genotypes (GM06890, GM20230, GM06906, and GM06852, respectively) were used to examine the analytic sensitivity of the TP-PCR with MCA assay. The amount of genomic DNA per test (reaction) recommended by the manufacturer is 50 ng. To determine the detection limit of the TP-PCR assay, we varied the amount of input DNA per reaction at 10, 25, 50, and 100 ng. We did not observe significant variations in the melt curve profiles with different amounts of DNA input (Figure 2A). Furthermore, the temperature at which baseline dF/dT temperature levels resumed was consistent across the range of DNA input levels for all four Coriell male DNA samples. With the lowest input of genomic DNA tested at 10 ng, melt curve profiles of the PM and FM genotypes remained clearly distinguishable from the NL sample. Similarly, the analytic sensitivity of the assay was tested with four Coriell female DNA samples (GM07538, GM20236, GM06906, and GM07537, respectively) representing distinct genotypes (Figure 2B). Although the melt curve profile of GM07538 at 100 ng had a lower signal, the corresponding resumed baseline dF/dT temperature was not altered significantly (Figure 2B). Regardless of DNA input amount, the resumed baseline dF/dT temperatures of the PM and FM samples were distinct from the NL and GZ samples. The results indicated that the TP-PCR assay with MCA has a detection limit of 10 ng (or lower) per test.

Analytic Specificity

On completion of the MCA, melt curve profiles were automatically generated with the negative first derivative of fluorescence plotted against temperature. Data were analyzed with software from the manufacturer of the real-time PCR platform, following the manufacturer’s instructions. Briefly,

The analytic specificity of the TP-PCR assay was assessed with a non-relevant DNA sample (GM23378) to gauge if nucleic acids distinct from CGG repeat expansions in FMR1 will affect kit performance. A Coriell male DNA sample (GM23378) that harbored a CTG repeat expansion in the dystrophia myotonica-protein kinase gene (DMPK) was added in increasing amounts (0, 50, 100, and 150 ng) to 50 ng each of four Coriell DNA samples, representing the female NL, GZ,

306

jmd.amjpathol.org

Data Analysis and Result Interpretation

-

The Journal of Molecular Diagnostics

Screening CGG Repeat Expansions in FMR1 expanded samples (GM06907 and GM07537) (Figure 3) even with 150 ng of potentially cross-reactive DNA (GM23378). The results indicated that the TP-PCR assay with MCA approach can differentiate expanded FMR1 alleles from nonexpanded alleles in the presence of other non-relevant DNA species.

Consistency According to the manufacturer’s recommendation, the TP-PCR assay required inclusion of an appropriate control sample to serve as a cutoff reference for the temperature at which the test sample resumes baseline dF/dT temperature. The consistency of this temperature parameter was evaluated with three Coriell DNA samples that are recommended controls for the TP-PCR assay. The number of FMR1 CGG repeats in male DNA samples GM06890, GM20244, and GM20230 are 30, 41, and 53 repeats, respectively. The resumed baseline dF/dT temperatures of these samples were obtained from a total of nine valid runs performed on the Rotor-Gene Q HRM system at two sites by two operators. The average resumed baseline dF/dT temperatures were distinct among the three samples (Figure 4A); replicates produced consistent results with CV < 0.5% (Figure 4B). We further tested the intrarun consistency of the resumed baseline dF/dT temperatures by using different recommended platforms, namely the ABI 7500 Fast Real-time PCR platform and the Qiagen Rotor-Gene Q HRM. We performed the assay by using the same three potential controls with 30, 41, and 53 CGG repeats. The average resumed baseline dF/dT temperatures were obtained for 96 replicates of each sample on the ABI 7500 Fast Real-time PCR platform and for 12 replicates of each sample on the Rotor-Gene Q HRM (Figure 4C and Table 2). The average resumed baseline dF/dT temperatures were consistent within the same run on each platform, giving a CV < 0.55% (Table 2). Figure 4

Melt curve profiles that show consistency of the resumed baseline dF/dT temperature as a parameter across PCR platforms. A: Melt curve profiles of potential cutoff controls: GM06890 (30 CGG repeats), GM20244 (41 CGG repeats), and GM20230 (53 CGG repeats) on the Rotor-Gene Q HRM platform. B: Average resumed baseline dF/dT temperatures of GM06890, GM20244, and GM20230 obtained on the Rotor-Gene Q HRM platform across nine valid runs operated independently at two sites. C: Intrarun average resumed baseline dF/dT temperatures of GM06890, GM20244, and GM20230 on the ABI and the Rotor-Gene Q HRM platforms. Data are expressed as means  SD. n Z 9 (B); n Z 96 (C, ABI); n Z 12 (C, RGQ). ABI, ABI 7500 Fast Real-Time PCR; resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed; RGQ, Rotor-Gene Q HRM platform.

PM, and FM genotypes (GM07538, GM20236, GM06907, and GM07537, respectively). With increasing amounts of nonrelevant DNA, the melt curve profiles of all four samples tested did not exhibit significant variation in terms of amplitude, shape, and resumed baseline dF/dT temperatures (Figure 3). Importantly, the NL and GZ samples (GM07538 and GM20236) produced a melt curve profile distinct from the

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

Detection of Mosaic Samples The performance of the TP-PCR assay with MCA in detecting mosaicism was initially evaluated with simulated samples and Table 2 Resumed Baseline dF/dT Temperatures of Controls Determined with Different PCR Platforms

Platform ABI 7500 Fast (n Z 96)

Rotor-Gene Q HRM (n Z 12)

CGG repeats, n

Average resumed baseline temperature,  C

CV, %

30 41 53 30 41 53

89.7 90.2 92.4 90.2 91.1 92.8

0.41 0.54 0.51 0.11 0.45 0.18

Resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed.

307

Lim et al

Figure 5

Melt curve profiles of simulated mosaic samples relative to a 53 CGG control (GM20230). Melt curve profiles of a male normal sample (GM06890) mixed with a male PM sample (GM06906) (A) or a male FM sample (GM06852) (B) in varying proportions while maintaining total DNA input at 50 ng. Resumed baseline dF/dT temperatures are compared with a 53 CGG control (GM20230) and a 30 CGG sample (GM06890). All reactions are performed in triplicates on the RotorGene Q HRM; representative profiles are shown. A: 100% to 7.5% PM mosaic samples are in the top panel, whereas 5% to 1% PM mosaic samples are in the lower panel. B: 100% to 20% FM mosaic samples are in the top panel, whereas 10% to 1% FM mosaic samples are in the lower panel. FM, full mutation; PM, premutation; resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed.

was subsequently verified with clinical samples (see Detection of Clinical Mosaic Samples). The simulated samples were generated by mixing a male normal sample (GM06890, 30 CGG repeats) with a male PM or FM sample (GM06906, 85 to 90 CGG repeats; GM06852, >200 CGG repeats). Samples were mixed in various proportions, yet maintained at a total DNA input of 50 ng per reaction. The simulated samples contained 1%, 2.5%, 5%, 7.5%, 10%, 20%, 50%, and 100% of the expanded sample. Melt curve profiles of the simulated samples were generated with the Rotor-Gene Q HRM and compared with that of a control sample harboring either 30 or 53 CGG repeats (Figure 5). We observed that the melt curve profiles of the 7.5% to 100% PM-simulated mosaic samples gave distinct and higher resumed baseline dF/dT temperatures than the 53 CGG repeat control (Figure 5A). The 2.5% and 5% PM mosaic samples had resumed baseline dF/dT temperatures that overlapped with

the 53 CGG repeat control and yet were distinct from the 30 CGG repeat normal sample (GM06890) (Figure 5A). We also observed that the melt curve profiles of the 20% to 100% FM mosaic samples gave higher resumed baseline dF/dT temperatures than the 53 CGG repeat control (Figure 5B). The 5% to 10% FM mosaic samples had resumed baseline dF/dT temperatures overlapping with the 53 CGG repeat control and yet were distinct from the 30 CGG repeat normal samples (GM06890) (Figure 5B). The results indicated that the TP-PCR assay with MCA detected expanded FMR1 alleles in the simulated mosaic samples at levels as low as 7.5% but no less than 20% when using a 53 CGG repeat control.

308

jmd.amjpathol.org

Kit Performance Evaluated with Reference Samples The performance of TP-PCR assay with MCA was evaluated in a blinded (G.X.Y.L.) study by using 35 Coriell

-

The Journal of Molecular Diagnostics

Screening CGG Repeat Expansions in FMR1 Table 3

Classification of Reference DNA Samples Using TP-PCR Assay with MCA in Comparison with Characterized Genotypes from Coriell Genotype from Coriell cell repositories (n Z 35)

Classification using TP-PCR þ MCA Cutoff control: GM20230 (53 CGG repeats) Nonexpanded (53 CGG repeats) Indeterminatey Expanded (>53 CGG repeats) Cutoff control: GM20244 (41 CGG repeats) Nonexpanded (41 CGG repeats) Indeterminatey Expanded (>41 CGG repeats) Cutoff control: GM06890 (30 CGG repeats) Nonexpanded (30 CGG repeats) Indeterminatey Expanded (>30 CGG repeats) Total samples per class, n

Normal (30 CGG repeats)

High normal (30e45 CGG repeats)

Gray zone (45e55 CGG repeats)

Pre-/Full mutation (55 CGG repeats)

6 0 0

1 0 0

2 0 1

1 0 24

94.2

6 0 0

0 0 1

0 0 3

0 0 25

97.1

2 1 3 6

0 0 1 1

0 0 3 3

0 0 25 25

Agreement, %*

88.6

*Percentage was determined on the basis of the fraction of samples undetected (nonexpanded) and detected (expanded and indeterminate) that was in agreement with the Coriell-reported genotypes with the different cutoff controls. y A sample classified as indeterminate warrants further testing and is considered a positive detection. MCA, melting curve analysis; TP-PCR, triplet repeat-primed PCR.

reference DNA samples on the Rotor-Gene Q HRM platform. The genotypes of the DNA samples tested are listed in Table 1. Three male samples with 30, 41, and 53 CGG repeats (GM06890, GM20244, and GM20230, respectively) were designated as cutoff reference controls for result interpretation. Our test results were summarized and compared with CGG repeat data provided by Coriell Cell Repositories (Table 3). As expected, the performance of the TP-PCR assay in detecting CGG repeat expansions depended on the choice of cutoff control (GM06890, GM20244, or GM20230) used for result interpretation. Samples classified as expanded have alleles of >30, >41, and >53 CGG repeats, respectively. The agreements between our test results and data provided by Coriell were 89%, 97%, and 94% when GM06890 (30 CGG repeats), GM20244 (41 CGG repeats), and GM20230 (53 CGG repeats) were used, respectively, as the cutoff control (Table 3). Notably, most of the disagreements derived from false detections by the assay, especially when GM06890 (30 CGG repeats) and GM20244 (41 CGG repeats) were used as the control (Table 3). The false detections arose from samples with CGG repeat lengths close to (within one repeat) those of the respective controls. The only disagreement due to a false negative when GM20230 (53 CGG repeats) was used as the control derived from a male sample with 56 CGG repeats, which marginally met the criterion for PM.

Kit Performance Validated with Clinical Samples The performance of the TP-PCR assay with MCA was ultimately evaluated in a blinded (G.X.Y.L. and Y.L.L.) and retrospective study by using genomic DNA derived from 528 patient whole blood samples from a population with intellectual disabilities.32 These clinical samples were

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

previously characterized for their FMR1 CGG repeat size by using a combination of flanking PCR,5 TP-PCR, and/or Southern Blot analysis.31 Again, three male samples from Coriell Cell Repositories with 30, 41, and 53 CGG repeats (GM06890, GM20244, and GM20230, respectively) were included in the blinded study and used as cutoff controls for result interpretation. The study was performed on the RotorGene Q HRM platform. The acquired data from the study were interpreted on the basis of the three cutoff reference controls (Table 4). With the 53 CGG repeat cutoff control (GM20230), the assay detected all 39 PM/FM samples (true positives), of which 38 were classified as expanded (>53 CGG repeats) and one was indeterminate (equal to 53 CGG repeats). Consequently, the detection of all PM/FM samples gave a clinical sensitivity of 100.00% (95% CI, 91.0%e100%) (Table 5). The assay also detected two of three GZ samples (false positives) in addition to the 39 PM/FM samples, resulting in a positive predictive value of 95.12%. Conversely, the assay scored as nonexpanded for all 370 NL samples, all 116 high normal samples, and one of three GZ samples (Table 4), resulting in a clinical specificity of 99.59% (95% CI, 98.5%e99.9%) (Table 5). None of the PM/FM alleles were scored as nonexpanded (false negatives), thereby giving a negative predictive value of 100.00% (Table 5). With the 41 CGG repeat cutoff control, expanded samples would comprise CGG repeats >41, and will include GZ alleles. Consequently, any GZ, PM, and FM sample that was undetected (scored as neither indeterminate nor expanded) would be considered as a false negative (Table 4). The TPPCR assay failed to detect one of three GZ samples, yet falsely detected 19 high normal samples, resulting in an overall sensitivity of 97.62% (95% CI, 87.7%e99.6%) and an overall specificity of 96.09% (95% CI, 93.8%e97.5%) (Table 5).

309

Lim et al Table 4 Classification of Clinical Samples Using the TP-PCR Assay with MCA in Comparison with Previously Characterized Methods from Mundhofir et al31 Classification from Mundhofir et al31

Classification using TP-PCR þ MCA Cutoff control: GM20230 (53 CGG repeats) Nonexpanded (53 CGG repeats) Indeterminate* Expanded (>53 CGG repeats) Cutoff control: GM20244 (41 CGG repeats) Nonexpanded (41 CGG repeats) Indeterminate* Expanded (>41 CGG repeats) Cutoff control: GM06890 (30 CGG repeats) Nonexpanded (30 CGG repeats) Indeterminate* Expanded (>30 CGG repeats) Total samples per class, n

Normal (30 CGG repeats)

High normal (30e45 CGG repeats)

Gray zone (45e55 CGG repeats)

Premutation/full mutation (55 CGG repeats)

370 0 0

116 0 0

1 2 0

0 1 38

370 0 0

97 10 9

1 0 2

0 0 39

301 31 38 370

10 4 102 116

0 0 3 3

0 0 39 39

*A sample classified as indeterminate warrants further testing and is considered a positive detection. MCA, melting curve analysis; TP-PCR, triplet repeat-primed PCR.

When the cutoff reference was extended to 30 CGG repeats, detection included high normal samples in addition to GZ, PM, and FM samples. Consequently, any sample classified as high normal, GZ, PM, and FM that was undetected (scored neither as indeterminate nor expanded) would be considered as a false negative (Table 4). On the basis of this criterion, the assay failed to detect 10 of 116 high normal samples, yet falsely detected 67 of the 370 NL samples. The performance of the assay in this case resulted in 93.67% sensitivity (95% CI, 88.7%e 96.5%) and 81.35% specificity (95% CI, 77.1%e85.0%) (Table 5).

Detection of Clinical Mosaic Samples Of the 528 clinical samples tested, three PM mosaic male samples harbored FMR1 alleles with CGG repeats of 86 of 166, 80 of 103, and 86 of 103 determined with other methods.5,31 These three samples were identified retrospectively on testing completion; respective melt curve profiles generated were specifically examined. The resumed baseline dF/dT temperatures of all three mosaic samples were clearly higher than that of the 53 CGG cutoff control (GM20230) (Figure 6), indicating detection of expanded alleles in clinical mosaic samples by using the TP-PCR assay. Table 5

Detection of Female Heterozygotes The study cohort included 139 female homozygous samples that carried two NL FMR1 alleles and 67 female heterozygotes that carried either two NL alleles of different sizes or one NL and one expanded allele. These samples were identified retrospectively on testing completion, and their data were specifically examined. The assay detected all female homozygous samples (n Z 139) with NL alleles and heterozygous samples that carried NL alleles of different sizes (n Z 44) as nonexpanded when the 53 CGG cutoff control (GM20230) was used (Table 6). Further, all such samples (n Z 139 þ 44) generated a resumed baseline dF/dT temperature that was much lower than that of the 53 CGG control (Figure 7). Conversely, all 23 female heterozygotes with PM and FM alleles were detected as expanded; their resumed baseline dF/dT temperatures were higher than that of the selected male 53 CGG control (GM20230). The temperature profiles of female heterozygotes with expanded alleles clearly differ from homozygous and heterozygous samples that contained only NL alleles (Figure 7).

Discussion The lack of a screening tool that is both rapid and costeffective for detecting FMR1 expansions in large populations

Clinical Performance of TP-PCR Assay with MCA Clinical performance, %

Cutoff control

Sensitivity (95% CI)

Specificity (95% CI)

PPV

NPV

53 CGG repeats 41 CGG repeats 30 CGG repeats

100.00 (91.0e100) 97.62 (87.7e99.6) 93.67 (88.7e96.5)

99.59 (98.5e99.9) 96.09 (93.8e97.5) 81.35 (77.1e85.0)

95.12 68.33 68.20

100.00 99.79 96.78

MCA, melting curve analysis; NPV, negative predictive value; PPV, positive predictive value; TP-PCR, triplet repeat-primed PCR.

310

jmd.amjpathol.org

-

The Journal of Molecular Diagnostics

Screening CGG Repeat Expansions in FMR1

Figure 6

Melt curve profiles of male mosaic clinical samples. Male mosaic samples from the clinical archive were detected through melt curve analysis on the Rotor-Gene Q HRM platform when a 53 CGG control (GM20230) was used. Resumed baseline dF/dT temperature cutoff refers to that of GM20230. NTC, no template control; resumed baseline dF/dT temperature, temperature at which baseline negative first derivative of fluorescence versus temperature resumed.

is well recognized.25 To tackle the problem, Tassone et al25 adopted an approach that combined two PCRs: first a standard flanking PCR followed by a second chimeric CGG repeat-targeted PCR. The first PCR could rapidly identify the normal male and the obvious heterozygous normal female, but a subsequent TP-PCR was required to resolve the ambiguity between a homozygous normal female and a female heterozygous for a non-amplified large expansion. Collectively, the two PCRs will reduce the need for the Southern blot testing.25 Other groups incorporated capillarybased methods into their TP-PCRs to increase throughput and to minimize reliance on Southern blot analysis.6,23 A third approach developed by Teo et al29 was unique, using MCA of TP-PCR amplicons to detect FMR1 CGG expansions. The combinatorial approach by Teo et al29 rapidly generates positive identification of PM and FM FMR1 alleles even in the presence of non-expanded alleles regardless of sex or zygosity. Such an approach contrasts with the method by Tassone et al25 which relies on a null-allele result for identifying expansions in males. Additional testing is also required for resolving ambiguity in females. Consequently, the TP-PCR with MCA approach by Teo et al29 reduces reliance on subsequent Southern blot analysis and also removes the requirement for post-PCR analysis via gel or capillary electrophoresis. In terms of equipment needs and reagent costs, the TP-PCR with MCA approach requires

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

minimally a real-time PCR platform, rather than an additional more expensive capillary electrophoresis instrument. Indeed, the TP-PCR with MCA approach is much more cost-effective and efficient and truly exemplifies a first-line PCR-only screening assay that requires no post-PCR processing. The FastFraX ID kit is a commercially available screening test developed using the TP-PCR approach with MCA. In our present work, we assessed the analytic performance of the assay by using reference DNA samples from the Coriell Cell Repositories. Importantly, we also evaluated its clinical performance in a blinded retrospective study with 528 clinical DNA samples from persons with intellectual disabilities; these DNA samples span a wide range of CGG repeat sizes. Our study verified that melt curve profiles generated by the TP-PCR with MCA approach distinguished expanded PM and FM samples from NL samples when the recommended 50 ng of DNA input was used (Figure 2). The robustness of the assay was demonstrated by the degree of clear separation between the melt curve profiles of expanded and the non-expanded samples, even when DNA inputs were as low as 10 ng per test (Figure 2). Melt curve profiles of the expanded and non-expanded profiles were maintained in the presence of 150 ng of non-relevant DNA (Figure 3). Further, the resumed baseline dF/dT temperatures of the melt curve profiles were consistent when the assay was validated with the ABI 7500 Fast and Rotor-Gene Q HRM. The former generated a CV < 0.55% and the latter 55 CGG repeats (eg, 30, 98, and 30, >200 CGG repeats) when used with a 53 CGG cutoff control. This finding indicated that the TP-PCR assay with MCA approach is unambiguous in differentiating heterozygous expanded samples with one NL and one expanded allele from homozygous NL samples (eg, 30, 30 CGG repeats, or two alleles of the same size or alleles differing by one to two repeats) (n Z 139) and heterozygous NL samples (n Z 44) (Table 6). Therefore, this test enables rapid screening for identification of all samples with at least one expanded FMR1 CGG repeat allele, without capillary electrophoresis or Southern blot analysis. Undoubtedly, this assay takes into consideration CGG repeat size but not methylation status, and so it is unable to provide information about skewed X-chromosome inactivation in females. TP-PCR is used to detect mosaic samples when coupled with capillary electrophoresis, by relying on a ladder motif generated.34 As such, detection of simulated mosaic samples with the combinatorial TP-PCR and MCA approach is unsurprising. A simulated mosaic sample with a PM allele present at 7.5% of total DNA input was detected when a 53 CGG cutoff control was used (Figure 5). The 53 cutoff control is stringent in detecting simulated samples; the 30 CGG control has a much lower resumed baseline dF/dT temperature and would have enabled the detection of lowlevel mosaics. Nevertheless, all three male mosaic samples in the clinical archive were detected with the stringent 53 CGG cutoff control (Figure 6). Although these clinical mosaic samples were easily identified because of the presence of two PM alleles, detection served as a confirmation for the utility of the assay. The TP-PCR assay with MCA approach appears to have its limitation in distinguishing PM from FM alleles because

The Journal of Molecular Diagnostics

-

jmd.amjpathol.org

of the overlap of resumed baseline dF/dT temperatures between the PM and FM samples. In theory, the resumed baseline dF/dT temperatures of the PM and FM amplicons should be distinguishable because the latter would contain longer amplicons due to larger alleles. In practice however, the assay is consistent with other TP-PCR approaches, producing only limited amounts of CGG amplicons beyond a certain size. As a result, the products of larger alleles are present in low proportions in the mixture of CGG amplicons. Thus, differences between the mixtures of amplicons that contain inconsequential amounts of a large and an even larger allele might not be detectable by MCA used in our approach. Nevertheless, our approach meets the intended utility for detecting samples with defined CGG expansions, markedly reducing the number of samples that require further characterization. Regardless, this screening assay will have to be complemented with additional approaches to precisely determine the CGG repeat size and FMR1 methylation status of the samples being interrogated. This MCA identification will be applicable also for other trinucleotide repeat disorder diseases.

References 1. Hill MK, Archibald AD, Cohen J, Metcalfe SA: A systematic review of population screening for fragile X syndrome. Genet Med 2010, 12: 396e410 2. Tassone F: Newborn screening for fragile X syndrome. JAMA Neurol 2014, 71:355e359 3. Pieretti M, Zhang F, Fu Y, Warren S, Oostra B, Caskey C, Nelson D: Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 1991, 66:817e822 4. Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP, et al: Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 1991, 65:905e914 5. Fu YH, Kuhl DP, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk AJ, Holden JJ, Fenwick RG Jr, Warren ST, et al: Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991, 67:1047e1058 6. Chen L, Hadd A, Sah S, Filipovic-Sadic S, Krosting J, Sekinger E, Pan R, Hagerman PJ, Stenzel TT, Tassone F, Latham GJ: An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis. J Mol Diagn 2010, 12:589e600 7. Hoffman GE, Le WW, Entezam A, Otsuka N, Tong ZB, Nelson L, Flaws JA, McDonald JH, Jafar S, Usdin K: Ovarian abnormalities in a mouse model of fragile X primary ovarian insufficiency. J Histochem Cytochem 2012, 60:439e456 8. Gallagher A, Hallahan B: Fragile X-associated disorders: a clinical overview. J Neurol 2012, 259:401e413 9. Hagerman P: Fragile X-associated tremor/ataxia syndrome (FXTAS): pathology and mechanisms. Acta Neuropathol 2013, 126:1e19 10. Monaghan KG, Lyon E, Spector EB; American College of Medical Genetics and Genomics: ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics. Genet Med 2013, 15: 575e586 11. de Vries B, Halley D, Oostra B, Niermeijer M: The fragile X syndrome. J Med Genet 1998, 35:579e589

313

Lim et al 12. Loesch D, Hagerman R: Unstable mutations in the FMR1 gene and the phenotypes. Adv Exp Med Biol 2012, 769:78e114 13. Cronister A, Teicher J, Rohlfs EM, Donnenfeld A, Hallam S: Prevalence and instability of fragile X alleles: implications for offering fragile X prenatal diagnosis. Obstet Gynecol 2008, 111:596e601 14. Nolin SL, Sah S, Glicksman A, Sherman SL, Allen E, Berry-Kravis E, Tassone F, Yrigollen C, Cronister A, Jodah M, Ersalesi N, Dobkin C, Brown WT, Shroff R, Latham GJ, Hadd AG: Fragile X AGG analysis provides new risk predictions for 45-69 repeat alleles. Am J Med Genet A 2013, 161A:771e778 15. Cronister A, Schreiner R, Wittenberger M, Amiri K, Harris K, Hagerman RJ: Heterozygous fragile X female: historical, physical, cognitive, and cytogenetic features. Am J Med Genet 1991, 38:269e274 16. Jacquemont S, Hagerman RJ, Leehey MA, Hall DA, Levine RA, Brunberg JA, Zhang L, Jardini T, Gane LW, Harris SW, Herman K, Grigsby J, Greco CM, Berry-Kravis E, Tassone F, Hagerman PJ: Penetrance of the fragile X-associated tremor/ataxia syndrome in a premutation carrier population. JAMA 2004, 291:460e469 17. Bourgeois JA, Seritan AL, Casillas EM, Hessl D, Schneider A, Yang Y, Kaur I, Cogswell JB, Nguyen DV, Hagerman RJ: Lifetime prevalence of mood and anxiety disorders in fragile X premutation carriers. J Clin Psychiatry 2011, 72:175e182 18. Grigsby J, Brega AG, Engle K, Leehey MA, Hagerman RJ, Tassone F, Hessl D, Hagerman PJ, Cogswell JB, Bennett RE, Cook K, Hall DA, Bounds LS, Paulich MJ, Reynolds A: Cognitive profile of fragile X premutation carriers with and without fragile X-associated tremor/ataxia syndrome. Neuropsychology 2008, 22:48e60 19. Hagerman R, Hagerman P: Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol 2013, 12:786e798 20. Loesch DZ, Tassone F, Lo J, Slater HR, Hills LV, Bui MQ, Silburn PA, Mellick GD: New evidence for, and challenges in, linking small CGG repeat expansion FMR1 alleles with Parkinson’s disease. Clin Genet 2013, 84:382e385 21. Murray J, Cuckle H, Taylor G, Hewison J: Screening for fragile X syndrome. Health Technol Assess 1997, 1:ieiv, 1-71 22. Oostra BA, Jacky PB, Brown WT, Rousseau F: Guidelines for the diagnosis of fragile X syndrome. National Fragile X Foundation. J Med Genet 1993, 30:410e413 23. Lyon E, Laver T, Yu P, Jama M, Young K, Zoccoli M, Marlowe N: A simple, high-throughput assay for Fragile X expanded alleles using

314

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

triple repeat primed PCR and capillary electrophoresis. J Mol Diagn 2010, 12:505e511 Filipovic-Sadic S, Sah S, Chen L, Krosting J, Sekinger E, Zhang W, Hagerman PJ, Stenzel TT, Hadd AG, Latham GJ, Tassone F: A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome. Clin Chem 2010, 56:399e408 Tassone F, Pan R, Amiri K, Taylor AK, Hagerman PJ: A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations. J Mol Diagn 2008, 10:43e49 Zhou Y, Law HY, Boehm CD, Yoon CS, Cutting GR, Ng IS, Chong SS: Robust fragile X (CGG)n genotype classification using a methylation specific triple PCR assay. J Med Genet 2004, 41:e45 Haddad LA, Mingroni-Netto RC, Vianna-Morgante AM, Pena SD: A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males. Hum Genet 1996, 97:808e812 Warner JP, Barron LH, Goudie D, Kelly K, Dow D, Fitzpatrick DR, Brock DJ: A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 1996, 33:1022e1026 Teo CR, Law HY, Lee CG, Chong SS: Screening for CGG repeat expansion in the FMR1 gene by melting curve analysis of combined 5’ and 3’ direct triplet-primed PCRs. Clin Chem 2012, 58:568e579 Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988, 16:1215 Mundhofir FE, Winarni TI, Nillesen WM, van Bon BW, Schepens M, Ruiterkamp-Versteeg M, Hamel BC, Yntema HG, Faradz SM: Prevalence of fragile X syndrome in males and females in Indonesia. World J Med Genet 2012, 2:15e22 Mundhofir FE, Winarni TI, van Bon BW, Aminah S, Nillesen WM, Merkx G, Smeets D, Hamel BC, Faradz SM, Yntema HG: A cytogenetic study in a large population of intellectually disabled Indonesians. Genet Test Mol Biomarkers 2012, 16:412e417 Liu Y, Winarni TI, Zhang L, Tassone F, Hagerman RJ: Fragile Xassociated tremor/ataxia syndrome (FXTAS) in grey zone carriers. Clin Genet 2013, 84:74e77 Juusola JS, Anderson P, Sabato F, Wilkinson DS, Pandya A, FerreiraGonzalez A: Performance evaluation of two methods using commercially available reagents for PCR-based detection of FMR1 mutation. J Mol Diagn 2012, 14:476e486

jmd.amjpathol.org

-

The Journal of Molecular Diagnostics