Abstract
X-chromosome inactivation occurs randomly in normal female cells. However, the inactivation can be skewed in patients with alterations in X-chromosome. In balanced X-autosome translocations, normal X is preferentially inactivated, while in unbalanced X alterations, the aberrant X is usually inactivated. Here, we present a novel strategy to verify the skewed X inactivation pattern through the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into cells, in 11 patients: five carriers of balanced X-autosome translocations and six of unbalanced X-chromosome alterations. Since EdU is a labeled nucleoside analog of thymidine, its incorporation during DNA synthesis can reveal late replication regions and the inactive X-chromosome. All EdU findings were validated by the human androgen receptor gene (HUMARA) assay. The late replication regions were easily and quickly visualized in all cells, where inactive Xs are marked with strong green fluorescence. It was observed that the normal X-chromosome was preferentially inactivated in patients with balanced X-autosome translocations; while the aberrant X-chromosome was inactivated in most cells from patients with unbalanced alterations. By performing the fluorescence-based EdU assay, the differences between the active and inactive X-chromosomes are more easily recognizable than by classic cytogenetic methods. Furthermore, EdU incorporation allows the observation of the late replication regions in autosomal segments present in X derivatives from X-autosome translocations. Therefore, EdU assay permits an accurate and efficient cytogenetic evaluation of the X inactivation pattern with a low-cost, easy to perform and highly reproducible technique.
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Acknowledgments
We thank PhD Dr. Andreas Houben, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), for his valuable suggestions of the establishment of the EdU assay protocol in our laboratory. This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
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L. Sisdelli and A. C. Vidi contributed equally to this work.
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439_2015_1622_MOESM1_ESM.tif
Supplementary material 1. Figure S1 Equation for calculating the percentage of alleles with major (CAG)n repeats (Psup). B represents the area value of the digested allele with the highest repeat units and, B’ represents the area value of the undigested allele with the highest repeat units. A represents the area value of the digested allele with the lowest repeat units and, A’ represents the area value of the undigested allele with the lowest repeat units (Bolduc et al. 2008). The X inactivation was considered random when the ratio presented values between 50 and 65 %. Ratio with values between 66 and 73 % was considered indeterminate. On the other hand, when the ratio presented value between 74 and 90 %, the XCI pattern was considered skewed. And finally, when the ratio was between 90 and 100 %, the X inactivation was considered extremely skewed. For the patient with three X-chromosomes, the percentage of the digested major allele was calculated combining the XCI pattern of the three alleles (major, minor and intermediate). (TIFF 25 kb)
439_2015_1622_MOESM2_ESM.tif
Supplementary material 2. Figure S2 Capillary electrophoresis from two controls and six patients carriers of X-chromosome alterations. a Undigested and digested male sample showing amplification in undigested sample only, suggesting the absence of the X inactivation. b Undigested and digested female sample showing amplification in both samples with similar peaks pattern between undigested and digested samples, suggesting a random X inactivation. c, d and e Patients with balanced X rearrangements, showing different peaks pattern between undigested and digested samples; c although the digested sample shows both allele peaks (222 bp and 228 bp), the allele 228 presents the highest peak, a typical skewed inactivation pattern; d, e digested samples show only the minor allele peaks (210 bp and 214 bp, respectively), a typical extremely skewed inactivation pattern, when this allele is inactive. f, g and h Patients with unbalanced X rearrangements, showing a different peaks pattern in the undigested and digested samples; f, g digested samples show only the major allele peaks (235 bp and 241 bp, respectively), a typical extremely skewed inactivation pattern, when this allele is inactive. f Patient 9, carrier of the unbalanced dic(X;15) karyotype, inherited two maternal alleles (214 bp and 235 bp) from the patient 3, carrier of the balanced dic(X;15) karyotype. Note that in his mother (patient 3), the inactive X presents the allele 214, while he presents the allele 235 in the inactive X. h Patient 11, inherited two maternal alleles (229 bp and 241 bp) from patient 10, however undigested and digested samples show similar peaks patterns, suggesting a random X inactivation between the three X-chromosomes. All amplicon size values are represented in base pairs (bp) at x-axis. (TIFF 2263 kb)
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Sisdelli, L., Vidi, A.C., Moysés-Oliveira, M. et al. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) as a novel strategy for identification of the skewed X inactivation pattern in balanced and unbalanced X-rearrangements. Hum Genet 135, 185–192 (2016). https://doi.org/10.1007/s00439-015-1622-x
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DOI: https://doi.org/10.1007/s00439-015-1622-x