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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

The molecular signatures of FSHD are complex, as illustrated by healthy and FSHD-type chromosomes. In the general healthy population, each chromosome 4q arm has a large polymorphic array of D4Z4 repeats containing more than 10 RUs. In FSHD1, there is a dominant contraction of one 4q array to between 1 and 10 D4Z4 repeat units, whereas FSHD2 is contraction-independent. There are two main allelic variants in the subtelomere distal to the array, termed A and B. A rare third classification of subtelomere, termed C, is used for subtelomeres that do not hybridize with probes for A or B due to distal sequence changes [18]. In some instances, the distal-most repeat fragment of the 4q D4Z4 array contains additional ~2 kb of D4Z4 sequence, resulting in a longer terminal RU in cis with a 4qA subtelomere; this type of 4qA allele is referred to as 4qA-L [15]. Both FSHD1 and FSHD2 are exclusively linked to the 4qA subtelomere allelic variants containing a PAS for the DUX4-fl mRNA [12, 15]. In addition, both FSHD1 and FSHD2 require the epigenetic disruption of the D4Z4 array to a less methylated and more relaxed chromatin state. Results of the described bisulfite sequencing assays are indicated by “+” if a bisulfite (BS) PCR product is produced and “–” if no BS PCR product is produced. *On rare occasions, due to primer degradation, a 10qA BS PCR product is detected; however, sequencing eliminates these from analysis. **Diagnosis of this healthy chromosome requires genomic PCR and sequencing of the 4qA subtelomere to identify a non-permissive 4qA PAS.
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Fig1: The molecular signatures of FSHD are complex, as illustrated by healthy and FSHD-type chromosomes. In the general healthy population, each chromosome 4q arm has a large polymorphic array of D4Z4 repeats containing more than 10 RUs. In FSHD1, there is a dominant contraction of one 4q array to between 1 and 10 D4Z4 repeat units, whereas FSHD2 is contraction-independent. There are two main allelic variants in the subtelomere distal to the array, termed A and B. A rare third classification of subtelomere, termed C, is used for subtelomeres that do not hybridize with probes for A or B due to distal sequence changes [18]. In some instances, the distal-most repeat fragment of the 4q D4Z4 array contains additional ~2 kb of D4Z4 sequence, resulting in a longer terminal RU in cis with a 4qA subtelomere; this type of 4qA allele is referred to as 4qA-L [15]. Both FSHD1 and FSHD2 are exclusively linked to the 4qA subtelomere allelic variants containing a PAS for the DUX4-fl mRNA [12, 15]. In addition, both FSHD1 and FSHD2 require the epigenetic disruption of the D4Z4 array to a less methylated and more relaxed chromatin state. Results of the described bisulfite sequencing assays are indicated by “+” if a bisulfite (BS) PCR product is produced and “–” if no BS PCR product is produced. *On rare occasions, due to primer degradation, a 10qA BS PCR product is detected; however, sequencing eliminates these from analysis. **Diagnosis of this healthy chromosome requires genomic PCR and sequencing of the 4qA subtelomere to identify a non-permissive 4qA PAS.

Mentions: Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent myopathy that indiscriminately affects males and females of all ages [1–3]. Although clinical muscle weakness typically manifests in the second or third decade of life, there is great variability in clinical severity, from a severe infantile form to individuals who remain asymptomatic throughout their lives [1, 2, 4–7]. Genetically, there are two classes of FSHD that are both linked to the chromosome 4q35 D4Z4 macrosatellite array (Figure 1). In the healthy population, these polymorphic regions exist as 11 or more repeat units (RUs) on each chromosome (24 to 35 RUs on average and up to ~120 [8]). A patient is genetically diagnosed with FSHD1 if pulse-field gel electrophoresis (PFGE) analysis indicates that one of the 4q alleles has a contraction in the D4Z4 array to below 11 RUs, while maintaining at least 1 RU, and the contraction is in cis with a FSHD-permissive 4A-type subtelomere containing a functional polyadenylation signal (PAS) for the pathogenic DUX4-fl (DUX4-full length) mRNA [9–15]. In contrast, the far less common form, FSHD2, is highly similar to FSHD1 in clinical presentation, yet it is contraction-independent and cannot be diagnosed or excluded by this common molecular diagnostic procedure [16, 17]. However, as with FSHD1, FSHD2 also requires a disease-permissive 4A-type subtelomere allele distal to the D4Z4 array on at least one 4q chromosome [15], suggesting the expression of DUX4-fl is likely a key mechanism in both forms of FSHD. Interestingly, the majority of 4A-type subtelomeres are, in fact, disease-permissive [15, 18]. FSHD1 and FSHD2 are also linked by epigenetic deregulation, typically assayed by DNA methylation analysis, of the 4qA FSHD-permissive allele [17, 19]. In healthy subjects, both copies of the 4q35 D4Z4 array as well as both copies of the 10q26 D4Z4 array have hypermethylated DNA (>35% CpGs assayed are methylated). In FSHD1 patients, the contracted 4q35 D4Z4 array exhibits DNA hypomethylation while the non-contracted 4q35 allele remains hypermethylated [17, 19, 20]. FSHD2 patients do not have contractions in either 4q35 array; however, both 4q35 D4Z4 arrays and both 10q26 D4Z4 arrays are severely hypomethylated (<25% CpGs assayed are methylated) due to mutations in the SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing1) gene, or other yet-to-be-identified epigenetic modifiers of D4Z4 repression [17, 19, 21]. These DNA hypomethylation signatures are specific to FSHD, as DNA methylation patterns of the 4q/10q D4Z4 arrays in other muscular dystrophies are similar to those found in healthy subjects [17].Figure 1


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)

The molecular signatures of FSHD are complex, as illustrated by healthy and FSHD-type chromosomes. In the general healthy population, each chromosome 4q arm has a large polymorphic array of D4Z4 repeats containing more than 10 RUs. In FSHD1, there is a dominant contraction of one 4q array to between 1 and 10 D4Z4 repeat units, whereas FSHD2 is contraction-independent. There are two main allelic variants in the subtelomere distal to the array, termed A and B. A rare third classification of subtelomere, termed C, is used for subtelomeres that do not hybridize with probes for A or B due to distal sequence changes [18]. In some instances, the distal-most repeat fragment of the 4q D4Z4 array contains additional ~2 kb of D4Z4 sequence, resulting in a longer terminal RU in cis with a 4qA subtelomere; this type of 4qA allele is referred to as 4qA-L [15]. Both FSHD1 and FSHD2 are exclusively linked to the 4qA subtelomere allelic variants containing a PAS for the DUX4-fl mRNA [12, 15]. In addition, both FSHD1 and FSHD2 require the epigenetic disruption of the D4Z4 array to a less methylated and more relaxed chromatin state. Results of the described bisulfite sequencing assays are indicated by “+” if a bisulfite (BS) PCR product is produced and “–” if no BS PCR product is produced. *On rare occasions, due to primer degradation, a 10qA BS PCR product is detected; however, sequencing eliminates these from analysis. **Diagnosis of this healthy chromosome requires genomic PCR and sequencing of the 4qA subtelomere to identify a non-permissive 4qA PAS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: The molecular signatures of FSHD are complex, as illustrated by healthy and FSHD-type chromosomes. In the general healthy population, each chromosome 4q arm has a large polymorphic array of D4Z4 repeats containing more than 10 RUs. In FSHD1, there is a dominant contraction of one 4q array to between 1 and 10 D4Z4 repeat units, whereas FSHD2 is contraction-independent. There are two main allelic variants in the subtelomere distal to the array, termed A and B. A rare third classification of subtelomere, termed C, is used for subtelomeres that do not hybridize with probes for A or B due to distal sequence changes [18]. In some instances, the distal-most repeat fragment of the 4q D4Z4 array contains additional ~2 kb of D4Z4 sequence, resulting in a longer terminal RU in cis with a 4qA subtelomere; this type of 4qA allele is referred to as 4qA-L [15]. Both FSHD1 and FSHD2 are exclusively linked to the 4qA subtelomere allelic variants containing a PAS for the DUX4-fl mRNA [12, 15]. In addition, both FSHD1 and FSHD2 require the epigenetic disruption of the D4Z4 array to a less methylated and more relaxed chromatin state. Results of the described bisulfite sequencing assays are indicated by “+” if a bisulfite (BS) PCR product is produced and “–” if no BS PCR product is produced. *On rare occasions, due to primer degradation, a 10qA BS PCR product is detected; however, sequencing eliminates these from analysis. **Diagnosis of this healthy chromosome requires genomic PCR and sequencing of the 4qA subtelomere to identify a non-permissive 4qA PAS.
Mentions: Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent myopathy that indiscriminately affects males and females of all ages [1–3]. Although clinical muscle weakness typically manifests in the second or third decade of life, there is great variability in clinical severity, from a severe infantile form to individuals who remain asymptomatic throughout their lives [1, 2, 4–7]. Genetically, there are two classes of FSHD that are both linked to the chromosome 4q35 D4Z4 macrosatellite array (Figure 1). In the healthy population, these polymorphic regions exist as 11 or more repeat units (RUs) on each chromosome (24 to 35 RUs on average and up to ~120 [8]). A patient is genetically diagnosed with FSHD1 if pulse-field gel electrophoresis (PFGE) analysis indicates that one of the 4q alleles has a contraction in the D4Z4 array to below 11 RUs, while maintaining at least 1 RU, and the contraction is in cis with a FSHD-permissive 4A-type subtelomere containing a functional polyadenylation signal (PAS) for the pathogenic DUX4-fl (DUX4-full length) mRNA [9–15]. In contrast, the far less common form, FSHD2, is highly similar to FSHD1 in clinical presentation, yet it is contraction-independent and cannot be diagnosed or excluded by this common molecular diagnostic procedure [16, 17]. However, as with FSHD1, FSHD2 also requires a disease-permissive 4A-type subtelomere allele distal to the D4Z4 array on at least one 4q chromosome [15], suggesting the expression of DUX4-fl is likely a key mechanism in both forms of FSHD. Interestingly, the majority of 4A-type subtelomeres are, in fact, disease-permissive [15, 18]. FSHD1 and FSHD2 are also linked by epigenetic deregulation, typically assayed by DNA methylation analysis, of the 4qA FSHD-permissive allele [17, 19]. In healthy subjects, both copies of the 4q35 D4Z4 array as well as both copies of the 10q26 D4Z4 array have hypermethylated DNA (>35% CpGs assayed are methylated). In FSHD1 patients, the contracted 4q35 D4Z4 array exhibits DNA hypomethylation while the non-contracted 4q35 allele remains hypermethylated [17, 19, 20]. FSHD2 patients do not have contractions in either 4q35 array; however, both 4q35 D4Z4 arrays and both 10q26 D4Z4 arrays are severely hypomethylated (<25% CpGs assayed are methylated) due to mutations in the SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing1) gene, or other yet-to-be-identified epigenetic modifiers of D4Z4 repression [17, 19, 21]. These DNA hypomethylation signatures are specific to FSHD, as DNA methylation patterns of the 4q/10q D4Z4 arrays in other muscular dystrophies are similar to those found in healthy subjects [17].Figure 1

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