Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome

Abstract

The life-threatening Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome is a genetically heterogeneous autosomal recessive disorder. Twenty percent of patients cannot be explained by mutations in the known ICF genes DNA methyltransferase 3B or zinc-finger and BTB domain containing 24. Here we report mutations in the cell division cycle associated 7 and the helicase, lymphoid-specific genes in 10 unexplained ICF cases. Our data highlight the genetic heterogeneity of ICF syndrome; however, they provide evidence that all genes act in common or converging pathways leading to the ICF phenotype.

Figure 1. Homozygous missense mutations in CDCA7 in five ICF3 patients.

(a) Schematic representation of CDCA7, with the identified homozygous missense mutations in red. Sequence outtake: 4-CXXC-type zinc-finger domain, CXXC motifs are underlined, mutated residues in red. (b–d) Sanger sequencing confirmation of missense mutations in CDCA7 in families A–C. All variants were homozygous, the reference sequence is displayed for comparison. (e) Sanger sequencing confirmation of the homozygous missense CDCA7 mutation in patients 2.2 (proband) and 2.3 of family D. Both parents are heterozygous for the variant, sibling 2.1 is unaffected and homozygous for the wild-type (wt) allele.

Figure 2. Mutations in HELLS in five ICF4 patients.

(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.