FSHD is linked to chromosomal 4q35 region, that contains a D4Z4 array of up to 200 units. The most common form, autosomal dominant FSHD1, is caused by a contraction of the 4q D4Z4 array to less than 11 units, whereas FSHD2 is caused by reduced levels of functional SMCHD1 protein (Structural maintenance of chromosomes flexible hinge domain-containing 1). Although with different mechanisms, both genetic defects lead to DNA hypomethylation at D4Z4 on 4qter causing chromatin relaxation. This genomic modulation provides a transcriptionally permissive chromatin environment that is associated with the expression of DUX4 gene, the best candidate FSHD gene, enclosed within each D4Z4 unit. DUX4 expression requires also the presence of a polyadenylation signal (PAS) distal to the last D4Z4 unit that stabilizes the DUX transcript. There are two different allelic forms of the region distal to the D4Z4 array, A and B. Although a D4Z4 array followed by an A "Telomere" is also present on 10q, a functional PAS sequence has been identified almost exclusively on 4qA alleles. Ultimately, vast majority of FSHD1 and FSHD2 subjects show hypomethylation at D4Z4 region associated with an A allele containing a functional PAS. Currently, FSHD diagnosis is based on the identification of shortened D4Z4 4q arrays (in FSHD1) or the presence of mutations in SMCHD1 (in FSHD2) and the assessment of the A/B genotype of 4q alleles and eventually by methylation analysis at the D4Z4 units. Our study aims at developing a new diagnostic test based on the methylation analysis of CpGs in the region distal to the D4Z4 array. Indeed, we considered that DNA methylation changes in this distal region may correlate with DUX4 expression more effectively than changes in the proximal units. Therefore, we evaluated the relationship between the DNA methylation, disease status, repeat size and clinical severity by analyzing CpGs located in the close proximity of the polyadenylation signal. We have been able to design bisulfite sequencing assays that allow the analysis of 10 CpGs in the close proximity of the PAS. Results using a 4qA PAS-specific assay in a group of 38 FSHD1, 14 FSHD2 and 18 control samples show significant differences between affected and unaffected subjects, supporting the potential usefulness of this assay as a diagnostic tool for FSHD diagnosis.
Development of a new methylation assay for FSHD diagnosis
Calandra P;Cascino I;Teveroni E;Galluzzi G;Moretti F;Deidda G
2014
Abstract
FSHD is linked to chromosomal 4q35 region, that contains a D4Z4 array of up to 200 units. The most common form, autosomal dominant FSHD1, is caused by a contraction of the 4q D4Z4 array to less than 11 units, whereas FSHD2 is caused by reduced levels of functional SMCHD1 protein (Structural maintenance of chromosomes flexible hinge domain-containing 1). Although with different mechanisms, both genetic defects lead to DNA hypomethylation at D4Z4 on 4qter causing chromatin relaxation. This genomic modulation provides a transcriptionally permissive chromatin environment that is associated with the expression of DUX4 gene, the best candidate FSHD gene, enclosed within each D4Z4 unit. DUX4 expression requires also the presence of a polyadenylation signal (PAS) distal to the last D4Z4 unit that stabilizes the DUX transcript. There are two different allelic forms of the region distal to the D4Z4 array, A and B. Although a D4Z4 array followed by an A "Telomere" is also present on 10q, a functional PAS sequence has been identified almost exclusively on 4qA alleles. Ultimately, vast majority of FSHD1 and FSHD2 subjects show hypomethylation at D4Z4 region associated with an A allele containing a functional PAS. Currently, FSHD diagnosis is based on the identification of shortened D4Z4 4q arrays (in FSHD1) or the presence of mutations in SMCHD1 (in FSHD2) and the assessment of the A/B genotype of 4q alleles and eventually by methylation analysis at the D4Z4 units. Our study aims at developing a new diagnostic test based on the methylation analysis of CpGs in the region distal to the D4Z4 array. Indeed, we considered that DNA methylation changes in this distal region may correlate with DUX4 expression more effectively than changes in the proximal units. Therefore, we evaluated the relationship between the DNA methylation, disease status, repeat size and clinical severity by analyzing CpGs located in the close proximity of the polyadenylation signal. We have been able to design bisulfite sequencing assays that allow the analysis of 10 CpGs in the close proximity of the PAS. Results using a 4qA PAS-specific assay in a group of 38 FSHD1, 14 FSHD2 and 18 control samples show significant differences between affected and unaffected subjects, supporting the potential usefulness of this assay as a diagnostic tool for FSHD diagnosis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
