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Identifying diagnostic DNA methylation profiles for facioscapulohumeral muscular dystrophy in blood and saliva using bisulfite sequencing.

Jones TI, Yan C, Sapp PC, McKenna-Yasek D, Kang PB, Quinn C, Salameh JS, King OD, Jones PL - Clin Epigenetics (2014)

Bottom Line: We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures.Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences.Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres.

View Article: PubMed Central - PubMed

Affiliation: The Wellstone Program & The Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA.

ABSTRACT

Background: Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin relaxation due to epigenetic changes at the 4q35 D4Z4 macrosatellite array. Molecular diagnostic criteria for FSHD are complex and involve analysis of high molecular weight (HMW) genomic DNA isolated from lymphocytes, followed by multiple restriction digestions, pulse-field gel electrophoresis (PFGE), and Southern blotting. A subject is genetically diagnosed as FSHD1 if one of the 4q alleles shows a contraction in the D4Z4 array to below 11 repeats, while maintaining at least 1 repeat, and the contraction is in cis with a disease-permissive A-type subtelomere. FSHD2 is contraction-independent and cannot be diagnosed or excluded by this common genetic diagnostic procedure. However, FSHD1 and FSHD2 are linked by epigenetic deregulation, assayed as DNA hypomethylation, of the D4Z4 array on FSHD-permissive alleles. We have developed a PCR-based assay that identifies the epigenetic signature for both types of FSHD, distinguishing FSHD1 from FSHD2, and can be performed on genomic DNA isolated from blood, saliva, or cultured cells.

Results: Samples were obtained from healthy controls or patients clinically diagnosed with FSHD, and include both FSHD1 and FSHD2. The genomic DNAs were subjected to bisulfite sequencing analysis for the distal 4q D4Z4 repeat with an A-type subtelomere and the DUX4 5' promoter region. We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures. DNA hypomethylation was restricted to the contracted 4qA chromosome in FSHD1 patients while healthy control subjects were hypermethylated. Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences. Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres.

Conclusions: We have developed an assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat. We show that the DNA methylation profile of saliva reflects FSHD status. This assay can distinguish FSHD from healthy controls, differentiate FSHD1 from FSHD2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.

No MeSH data available.


Related in: MedlinePlus

Bisulfite (BS) PCRs using genomic DNAs from subjects with a range of 4q allelic combinations show the specificity of the three bisulfite sequencing (BSS) assays. Nested PCRs were performed using BS-converted genomic DNAs from seven subjects, five FSHD1 and two healthy, with varying 4q haplotypes (4qA/A, 4qA/B, 4qB/B, and 4qA/A-L, as indicated). The primer sets used are indicated to the right of each panel. The 4qA BS PCR (upper panel) amplified a product from all five subjects possessing at least 1 4qA allele and did not amplify any detectable product from the two subjects lacking a 4qA allele. The 4qA-L BS PCR (middle panel) only amplified a product from the one subject possessing a 4qA-L allele. The DUX4 5’ BS PCR (lower panel) amplified a product from all seven subjects. The identities of all BS PCR products were confirmed by sequencing.
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Fig3: Bisulfite (BS) PCRs using genomic DNAs from subjects with a range of 4q allelic combinations show the specificity of the three bisulfite sequencing (BSS) assays. Nested PCRs were performed using BS-converted genomic DNAs from seven subjects, five FSHD1 and two healthy, with varying 4q haplotypes (4qA/A, 4qA/B, 4qB/B, and 4qA/A-L, as indicated). The primer sets used are indicated to the right of each panel. The 4qA BS PCR (upper panel) amplified a product from all five subjects possessing at least 1 4qA allele and did not amplify any detectable product from the two subjects lacking a 4qA allele. The 4qA-L BS PCR (middle panel) only amplified a product from the one subject possessing a 4qA-L allele. The DUX4 5’ BS PCR (lower panel) amplified a product from all seven subjects. The identities of all BS PCR products were confirmed by sequencing.

Mentions: Dramatic epigenetic differences at the 4q35 D4Z4 repeat array between healthy and disease states distinguish FSHD1 and FSHD2 from unaffected individuals. Epigenetic differences at the non-contracted 4q35 D4Z4 and the 10q26 D4Z4 arrays distinguish FSHD2 from FSHD1 and other myopathies. In all forms of FSHD, it is the distal 4q35 D4Z4 in cis with a disease-permissive 4A subtelomere that produces the pathogenic DUX4-fl mRNA [15]. However, this pathogenic D4Z4 repeat has never been specifically analyzed in FSHD1 or FSHD2 [17, 19, 20, 29]. Therefore, in order to study epigenetic changes at the disease-relevant D4Z4 repeat, we developed two BSS assays that specifically analyze the distal 4qA- or 4qA-L-associated D4Z4 RU (Figure 2). Utilizing polymorphisms in the BSS PCR primers that are exclusive to the disease-permissive 4A subtelomere and not found in 10A, the 4qA BSS assay analyzes the DNA methylation status of 56 CpGs (Additional file 1: Figure S1A) in the distal D4Z4 RU in cis with a 4A subtelomere (Figure 2B). The 4qA bisulfite (BS)-PCR product is amplified from all BS-converted genomic DNAs from subjects possessing at least one 4qA allele (Figure 3, upper panel). The D4Z4-4A fragments were sequenced and, importantly, all 56 CpGs predicted by the reference sequence were accounted for in >90% of the analyzed sequences from these clones, confirming the specificity of the reaction for the distal 4qA-derived D4Z4. The 4qA-L BSS assay utilizes the same 4A subtelomere-specific reverse BS PCR primers as above; however, these are paired with a 4qA-L-specific forward BS PCR primer. The 4qA-L BSS assay analyzes the DNA methylation status of 30 CpGs (Additional file 1: Figure S1D) in the distal D4Z4 repeat on 4qA-L chromosomes (Figure 2C). The 4qA-L BS PCR product was amplified exclusively from BS-converted genomic DNAs from the one subject possessing a 4qA-L allele and not from any of the six subjects lacking a 4qA-L allele (Figure 3, middle panel). The 4qA-L fragment was sequenced and all 30 CpGs predicted by the reference sequence were accounted for in 100% of the analyzed sequences from these clones, confirming the specificity of the reaction for the distal 4qA-L-derived D4Z4. Neither the 4qA BS PCR nor the 4qA-L BS PCR produced a product from genomic DNAs isolated from either of the two healthy subjects with 4qB/B haplotypes. It is worth noting that due to the 4qA-specific SNPs residing at the 3’ end of the 4qA BS PCR oligonucleotide primers, multiple rounds of primer freeze-thaw, which leads to partial primer degradation, results in a loss of specificity and a consequent amplification of minor products from genomic DNAs lacking the 4qA allele (Additional file 1: Figure S1B). Sequence analysis of these rare amplicons from 4qB/B samples identified them as either a 10qA product (Additional file 1: Figure S1C) or a non-specific product not derived from any D4Z4 or 4qA/B allelic variant. In addition, since the BSS analysis is sequence-based and not product-based (as in qPCR or Southern blotting), any rare non-specific or 10qA amplifications present are easily identified and removed from the analysis. We conclude that these assay conditions amplify the distal D4Z4 sequence from 4qA chromosomes or 4qA-L chromosomes, depending on the assay, and neither assay amplifies 10A or 4B subtelomere-containing chromosomes.Figure 3


Identifying diagnostic DNA methylation profiles for facioscapulohumeral muscular dystrophy in blood and saliva using bisulfite sequencing.

Jones TI, Yan C, Sapp PC, McKenna-Yasek D, Kang PB, Quinn C, Salameh JS, King OD, Jones PL - Clin Epigenetics (2014)

Bisulfite (BS) PCRs using genomic DNAs from subjects with a range of 4q allelic combinations show the specificity of the three bisulfite sequencing (BSS) assays. Nested PCRs were performed using BS-converted genomic DNAs from seven subjects, five FSHD1 and two healthy, with varying 4q haplotypes (4qA/A, 4qA/B, 4qB/B, and 4qA/A-L, as indicated). The primer sets used are indicated to the right of each panel. The 4qA BS PCR (upper panel) amplified a product from all five subjects possessing at least 1 4qA allele and did not amplify any detectable product from the two subjects lacking a 4qA allele. The 4qA-L BS PCR (middle panel) only amplified a product from the one subject possessing a 4qA-L allele. The DUX4 5’ BS PCR (lower panel) amplified a product from all seven subjects. The identities of all BS PCR products were confirmed by sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232706&req=5

Fig3: Bisulfite (BS) PCRs using genomic DNAs from subjects with a range of 4q allelic combinations show the specificity of the three bisulfite sequencing (BSS) assays. Nested PCRs were performed using BS-converted genomic DNAs from seven subjects, five FSHD1 and two healthy, with varying 4q haplotypes (4qA/A, 4qA/B, 4qB/B, and 4qA/A-L, as indicated). The primer sets used are indicated to the right of each panel. The 4qA BS PCR (upper panel) amplified a product from all five subjects possessing at least 1 4qA allele and did not amplify any detectable product from the two subjects lacking a 4qA allele. The 4qA-L BS PCR (middle panel) only amplified a product from the one subject possessing a 4qA-L allele. The DUX4 5’ BS PCR (lower panel) amplified a product from all seven subjects. The identities of all BS PCR products were confirmed by sequencing.
Mentions: Dramatic epigenetic differences at the 4q35 D4Z4 repeat array between healthy and disease states distinguish FSHD1 and FSHD2 from unaffected individuals. Epigenetic differences at the non-contracted 4q35 D4Z4 and the 10q26 D4Z4 arrays distinguish FSHD2 from FSHD1 and other myopathies. In all forms of FSHD, it is the distal 4q35 D4Z4 in cis with a disease-permissive 4A subtelomere that produces the pathogenic DUX4-fl mRNA [15]. However, this pathogenic D4Z4 repeat has never been specifically analyzed in FSHD1 or FSHD2 [17, 19, 20, 29]. Therefore, in order to study epigenetic changes at the disease-relevant D4Z4 repeat, we developed two BSS assays that specifically analyze the distal 4qA- or 4qA-L-associated D4Z4 RU (Figure 2). Utilizing polymorphisms in the BSS PCR primers that are exclusive to the disease-permissive 4A subtelomere and not found in 10A, the 4qA BSS assay analyzes the DNA methylation status of 56 CpGs (Additional file 1: Figure S1A) in the distal D4Z4 RU in cis with a 4A subtelomere (Figure 2B). The 4qA bisulfite (BS)-PCR product is amplified from all BS-converted genomic DNAs from subjects possessing at least one 4qA allele (Figure 3, upper panel). The D4Z4-4A fragments were sequenced and, importantly, all 56 CpGs predicted by the reference sequence were accounted for in >90% of the analyzed sequences from these clones, confirming the specificity of the reaction for the distal 4qA-derived D4Z4. The 4qA-L BSS assay utilizes the same 4A subtelomere-specific reverse BS PCR primers as above; however, these are paired with a 4qA-L-specific forward BS PCR primer. The 4qA-L BSS assay analyzes the DNA methylation status of 30 CpGs (Additional file 1: Figure S1D) in the distal D4Z4 repeat on 4qA-L chromosomes (Figure 2C). The 4qA-L BS PCR product was amplified exclusively from BS-converted genomic DNAs from the one subject possessing a 4qA-L allele and not from any of the six subjects lacking a 4qA-L allele (Figure 3, middle panel). The 4qA-L fragment was sequenced and all 30 CpGs predicted by the reference sequence were accounted for in 100% of the analyzed sequences from these clones, confirming the specificity of the reaction for the distal 4qA-L-derived D4Z4. Neither the 4qA BS PCR nor the 4qA-L BS PCR produced a product from genomic DNAs isolated from either of the two healthy subjects with 4qB/B haplotypes. It is worth noting that due to the 4qA-specific SNPs residing at the 3’ end of the 4qA BS PCR oligonucleotide primers, multiple rounds of primer freeze-thaw, which leads to partial primer degradation, results in a loss of specificity and a consequent amplification of minor products from genomic DNAs lacking the 4qA allele (Additional file 1: Figure S1B). Sequence analysis of these rare amplicons from 4qB/B samples identified them as either a 10qA product (Additional file 1: Figure S1C) or a non-specific product not derived from any D4Z4 or 4qA/B allelic variant. In addition, since the BSS analysis is sequence-based and not product-based (as in qPCR or Southern blotting), any rare non-specific or 10qA amplifications present are easily identified and removed from the analysis. We conclude that these assay conditions amplify the distal D4Z4 sequence from 4qA chromosomes or 4qA-L chromosomes, depending on the assay, and neither assay amplifies 10A or 4B subtelomere-containing chromosomes.Figure 3

Bottom Line: We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures.Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences.Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres.

View Article: PubMed Central - PubMed

Affiliation: The Wellstone Program & The Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA.

ABSTRACT

Background: Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin relaxation due to epigenetic changes at the 4q35 D4Z4 macrosatellite array. Molecular diagnostic criteria for FSHD are complex and involve analysis of high molecular weight (HMW) genomic DNA isolated from lymphocytes, followed by multiple restriction digestions, pulse-field gel electrophoresis (PFGE), and Southern blotting. A subject is genetically diagnosed as FSHD1 if one of the 4q alleles shows a contraction in the D4Z4 array to below 11 repeats, while maintaining at least 1 repeat, and the contraction is in cis with a disease-permissive A-type subtelomere. FSHD2 is contraction-independent and cannot be diagnosed or excluded by this common genetic diagnostic procedure. However, FSHD1 and FSHD2 are linked by epigenetic deregulation, assayed as DNA hypomethylation, of the D4Z4 array on FSHD-permissive alleles. We have developed a PCR-based assay that identifies the epigenetic signature for both types of FSHD, distinguishing FSHD1 from FSHD2, and can be performed on genomic DNA isolated from blood, saliva, or cultured cells.

Results: Samples were obtained from healthy controls or patients clinically diagnosed with FSHD, and include both FSHD1 and FSHD2. The genomic DNAs were subjected to bisulfite sequencing analysis for the distal 4q D4Z4 repeat with an A-type subtelomere and the DUX4 5' promoter region. We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures. DNA hypomethylation was restricted to the contracted 4qA chromosome in FSHD1 patients while healthy control subjects were hypermethylated. Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences. Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres.

Conclusions: We have developed an assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat. We show that the DNA methylation profile of saliva reflects FSHD status. This assay can distinguish FSHD from healthy controls, differentiate FSHD1 from FSHD2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.

No MeSH data available.


Related in: MedlinePlus