X-chromosomal insertions at a recurrent site causing ectodermal dysplasia

ARTICLE

Auteur(s) : Holm Schneider¹, Christiane Mühle²

¹Children’s Hospital, University of Erlangen-Nuernberg, Germany
²Department of Pediatrics, Medical University of Innsbruck, Innrain 66, 6020 Innsbruck, Austria

X-linked hypohidrotic ectodermal dysplasia (XLHED; Christ-Siemens-Touraine syndrome; MIM 305100), the most common form of ectodermal dysplasia, is caused by mutations of the gene EDA (previously ED1), encoding ectodysplasin-A. Recently, Tariq et al. [1] reported in this journal a 4bp insertion mutation (c.913_914insTATA) in EDA and suggested slipped-strand mispairing as the mechanism for this duplication of the original TATA sequence. Here, we describe another novel insertion at the same place and elucidate how slipped-strand mispairing during DNA replication can cause such insertions.

Genomic DNA of a boy with characteristic features of XLHED (figure 1A), e.g. sparse hair, saddle nose, conical or missing teeth, inability to sweat and recurrent fever, was investigated after informed consent. Portions of the X-chromosomal EDA including all exons and intron-exon boundaries were amplified by polymerase chain reaction and sequenced using the BigDye Terminator mix (Applied Biosystems) and a capillary sequencer. In exon 8 of EDA (NM_001399, longest transcript), we detected an insertion of two nucleotides (c.913_914insTA; figures 1B and C). It was present heterozygously in the patient’s mother (figure 1D), but absent in the father and control individuals (figure 1E). The mutation results in a frameshift leading to a premature termination codon three nucleotide triplets downstream (p.Ser305IlefsX4). The mutant mRNA is therefore predicted to be degraded by nonsense-mediated decay or to produce a truncated ectodysplasin A1 protein lacking 87 C-terminal amino acids. Loss of more than half of the TNF homology domain (amino acids 250-391) is likely to affect not only the overall structure of this protein but also its interaction with the ectodysplasin receptor. The mutation has not yet been reported in the literature and is not listed in the accessible part of the Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff.

The X-chromosome is particularly prone to insertions and deletions due to the flexibility of its telomeric end in the absence of a pairing X-chromosome during male meiosis [2]. Further sequence analysis attempting to elucidate the mechanism of insertional mutagenesis at this recurrent site revealed neither repeat elements (e.g. SINE, LINE, LTR) nor CpG islands in its vicinity (± 500 bp). No pseudogenes or genes with sequence similarity to the mutated DNA region, which may have promoted mutagenesis by gene conversion, were found.

Thus, the mutation in our patient’s family is likely to represent a TA repeat expansion at the same location as reported by Tariq et al. for an unrelated family with an expansion by two TA units (figure 1B). Repeat sequences are known for their susceptibility to slipped-strand mispairing. During DNA replication, slippage between template and primer strands may produce a loop of one unpaired repeat unit in the primer strand resulting in a misaligned intermediate that is stabilized by correct base pairing of the next unit and addition of nucleotides (TA or TATA, respectively) upon subsequent polymerization. Such slippage can occur either in the active site of the DNA polymerase or prior to substrate binding by the enzyme [3]. Studies in E. coli [4] showed that self-complementary sequence repeats (GpC, ApT) are the most unstable. Rubinsztein et al. [5] described a significant trend towards increased microsatellite length in humans, suggesting that their evolution is directional and faster than in other species. Among other hypotheses, these authors speculate that genetic modification of a polymerase or a mismatch repair enzyme might be responsible for the higher mutation rate.

Our finding and analysis of a novel insertion mutation may thus corroborate and explain the previously inscrutable assumption of Tariq et al. that slipped-strand mispairing may cause insertions at position 913_914 of EDA in analogy to repeat expansions for microsatellite loci.

Acknowledgements

References

2 Mühle C, Zenker M, Chuzhanova N, Schneider H. Recurrent inversion with concomitant deletion and insertion events in the coagulation factor VIII gene suggests a new mechanism for X-chromosomal rearrangements causing hemophilia A. Hum Mutat 2007; 28: 1045; (e1-7).

3 Kunkel TA, Bebenek K. DNA replication fidelity. Annu Rev Biochem 2000; 69: 497-529.

4 Bichara M, Pinet I, Schumacher S, Fuchs RP. Mechanisms of dinucleotide repeat instability in Escherichia coli. Genetics 2000; 154: 533-42.

5 Rubinsztein DC, Amos W, Leggo J, et al. Microsatellite evolution - evidence for directionality and variation in rate between species. Nat Genet 1995; 10: 337-43.