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In a 4qA/A Fsubjectbject for whom all chromosomes with the contracted array have lower 4qA BSS methylation than any chromosomes with the non-contracted array, Q1 gives an estimate of the median 4qA methylation of just the contracted array.
In myoblasts from one FSHD patient, higher levels of D4Z4 derived RNA could be detected [26], which may reflect stabilized transcripts originating from the most distal edge of the contracted array [27].
Moreover, 4q35.2 D4Z4 repeats are methylated in the general population, whereas the contracted array is hypomethylated in FSHD patients [ 6, 7].
A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.
In FSHD1 subjects with 4qA/A or 4qA-L/A-L haplotypes, on average half of the analyzed chromosomes will be derived from the contracted array and are expected to show DNA hypomethylation while the other half will be derived from the non-contracted array and are expected to show hypermethylation.
These polymorphic FSHD1-sized D4Z4 contractions by themselves are not pathogenic, and development of FSHD also requires a disease-permissive allele of the chromosome 4q subtelomere (4A) in cis with the contracted array [ 14- 17].
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By contrast, FSHD1 subjects should contain a combination of hypermethylated (from D4Z4 RUs residing in the non-contracted 4q array and both 10q arrays) and hypomethylated (from the D4Z4 RUs residing in the contracted 4q array) sequences with a clear minority of the analyzed D4Z4 RUs being hypomethylated; FSHD2 subjects should be hypomethylated (~ <25% methylation) on most sequences analyzed.
Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.
In FSHD1 patients, the contracted 4q35 D4Z4 array exhibits DNA hypomethylation while the non-contracted 4q35 allele remains hypermethylated [ 17, 19, 20].
In FSHD1 subjects with 4qA/B haplotypes, all of the analyzed chromosomes are derived from the contracted D4Z4 array and are expected to show hypomethylation.
To test this, we used cells isolated from FSHD1 (54-2, which are isogenic to normal 54-1 cells but carry a contracted D4Z4 array) and FSHD2 (MB200) skeletal muscle (Krom et al., 2012; Schoenberg and Maquat, 2012; Young et al., 2013).
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