Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1

Abstract

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95 bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Keywords: ANAPC1; RECQL4; Rothmund-Thomson syndrome; alternative splicing; anaphase-promoting complex; cataracts; cryptic splice site; poikiloderma; pseudoexon; splicing variant.

Figure 1

ANAPC1 Mutations in Individuals with Rothmund-Thomson Syndrome Type 2 (A) Photos of three affected individuals show the alopecia and abnormal teeth, nails, and skin. (B, top) Pedigrees of ten individuals from seven families with RTS type 1 and ANAPC1 mutations. All individuals affected have the ANAPC1 intronic splicing variant (GenBank: NM_022662.3:c.[2705−198C>T]). Individuals 1, 2, 3A, 3B, and 4 are homozygous for the ANAPC1 intronic splicing variant, whereas individuals 5, 6A, 6B, 7A, and 7B have compound-heterozygous mutations. (B, bottom) Graphical representation of all ANAPC1 variants identified. Introns other than 22 are not drawn to scale. The domains of ANAPC1 are discussed in detail by Li et al. and Chang et al., The WD40 domain mediates conformational changes important for stimulating the APC’s catalytic activity upon co-activator binding. The Mid-N and Mid-C domains coalesce together in the APC complex. The PC domain joins the APC platform to the tetratricopeptide repeat lobe of the APC.

Figure 2

Effect of the ANAPC1 Splicing Variant on Protein Levels and the Mechanism of Pseudoexon Activation in Fibroblasts Derived from Individuals 1 and 2 (A) Realtime PCR analysis demonstrating significant difference (p < 0.001) in messenger RNA from individuals 1 and 2 compared to unrelated controls. (B) An immunoblot showing a decrease in ANAPC1 protein levels in fibroblasts from individuals 1 and 2 compared to unrelated controls. (C) Realtime PCR with primers spanning exons 22 and 23 of ANAPC1 and subsequent agarose gel electrophoresis revealing the presence of an additional fragment in fibroblasts from affected individuals. Sanger sequencing of the additional fragment revealed the presence of a 95 bp nucleotide that is incorporated between exon 22 and 23 of ANAPC1 cDNA. Analysis of the pseudoexon revealed two stop codons leading to the nonsense-mediated decay pathway, accounting for the decrease in RTS type 1 individual transcript (A) and protein (B) levels.

Figure 3

Effect of ANAPC1 Deficiency and Cell Cycle Variations of RECQL4 (A) Fibroblasts derived from individuals homozygous for the ANAPC1 intronic variant spent a longer time at the interphase of the cell cycle compared to control fibroblasts (^∗∗∗ = p < 0.001); the time-lapse microscopy was performed as described previously. The middle line represents the median, the box represents quartiles, and the whiskers represent minimum and maximum values within 1.5 times the interquartile range. (B) Proteins from thymidine-nocodazole-synchronized-MG132-treated and non-treated HeLa cells at specific time points (0 to 8 hours) were analyzed by SDS-PAGE and immunoblot. The upward-shifted (phospho) APC3 represents a marker for mitosis. The cyclin B disappearance represents a positive control for APC/C activity, and actin is used as a loading control. RECQL4 protein levels decreased within the first 3 h of mitosis and became stable after treatment with MG132, similar to classical APC/C substrates such as cyclin B. Asyn signifies asynchronous cells. (C) Mice heterozygous for a loss-of-function Anapc1 mutation develop cataracts more frequently than wild-type mice (detailed data: no lens opacity in 2,766 WT (wild-type) mice and 12 het (heterozygous KO) mice, lens opacity in one eye in 116 WT mice and 4 het mice, lens opacities in both eyes in 17 WT mice and no het mice).