Figure 2: Mutations in HELLS in five ICF4 patients. | Nature Communications

Figure 2: Mutations in HELLS in five ICF4 patients.

From: Mutations in CDCA7 and HELLS cause immunodeficiency–centromeric instability–facial anomalies syndrome

Figure 2

(a) Schematic representation of HELLS, with the identified mutations in red. (b) Sanger sequencing confirmation of HELLS mutations in family E. Only c.370+2T>A was identified in maternal DNA, indicating different allelic origins of both mutations, or de novo occurrence of the second mutation. (c) RT–PCR analysis of HELLS mRNA on treatment of patient-derived fibroblasts with cycloheximide (C) revealed that c.370+2T>A leads to complete skipping of exon 5 and disruption of the open reading frame. Ethanol-treated samples (E) served as controls, alternative splicing was confirmed using Sanger’s sequencing in two independent experiments for both samples. (d) Sanger sequencing confirmation of a homozygous out-of-frame deletion in HELLS in family F. Both parents as well as unaffected sibling 2.1 are heterozygous for the deletion allele; unaffected sibling 2.3 is homozygous for the wt allele. (e) Sanger sequencing confirmation of a homozygous in-frame deletion in HELLS in family G. Both parents are heterozygous for the deletion allele. (f) Sanger sequencing confirmation of nonsense mutations in HELLS in family H. Different allelic origin was confirmed in parental DNA. (g) Southern blot analysis of minor satellite DNA methylation in Dnmt3b−/− and siRNA-treated wt MEFs after digesting DNA with MspI or its methylation-sensitive isoschizomer HpaII revealed CpG hypomethylation on knockdown of Zbtb24, Cdca7 and Hells. Molecular weights of the 2-Log DNA size marker are in kilobasepairs.

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