Research Article: Exome chip association study excluded the involvement of rare coding variants with large effect sizes in the etiology of anorectal malformations

Date Published: May 28, 2019

Publisher: Public Library of Science

Author(s): Romy van de Putte, Charlotte H. W. Wijers, Heiko Reutter, Sita H. Vermeulen, Carlo L. M. Marcelis, Erwin Brosens, Paul M. A. Broens, Markus Homberg, Michael Ludwig, Ekkehart Jenetzky, Nadine Zwink, Cornelius E. J. Sloots, Annelies de Klein, Alice S. Brooks, Robert M. W. Hofstra, Sophie A. C. Holsink, Loes F. M. van der Zanden, Tessel E. Galesloot, Paul Kwong-Hang Tam, Marloes Steehouwer, Rocio Acuna-Hidalgo, Maartje van de Vorst, Lambertus A. Kiemeney, Maria-Mercè Garcia-Barceló, Ivo de Blaauw, Han G. Brunner, Nel Roeleveld, Iris A. L. M. van Rooij, Mathias Toft.


Anorectal malformations (ARM) are rare congenital malformations, resulting from disturbed hindgut development. A genetic etiology has been suggested, but evidence for the involvement of specific genes is scarce. We evaluated the contribution of rare and low-frequency coding variants in ARM etiology, assuming a multifactorial model.

We analyzed 568 Caucasian ARM patients and 1,860 population-based controls using the Illumina HumanExome Beadchip array, which contains >240,000 rare and low-frequency coding variants. GenomeStudio clustering and calling was followed by re-calling of ‘no-calls’ using zCall for patients and controls simultaneously. Single variant and gene-based analyses were performed to identify statistically significant associations, applying Bonferroni correction. Following an extra quality control step, candidate variants were selected for validation using Sanger sequencing.

When we applied a MAF of ≥1.0%, no variants or genes showed statistically significant associations with ARM. Using a MAF cut-off at 0.4%, 13 variants initially reached statistical significance, but had to be discarded upon further inspection: ten variants represented calling errors of the software, while the minor alleles of the remaining three variants were not confirmed by Sanger sequencing.

Our results show that rare and low-frequency coding variants with large effect sizes, present on the exome chip do not contribute to ARM etiology.

Partial Text

Congenital anorectal malformations (ARM) are the most frequent malformations of the gastrointestinal tract, with a prevalence of 2 to 7 in 10,000 live births[1]. ARM encompass a broad range of phenotypes, which are usually classified according to the type of fistula to neighboring organs. In approximately 50% of the patients, ARM is associated with additional congenital malformations, including vertebral, cardiac and/or renal malformations[2, 3]. Multiple surgical corrections are required during the first years of a patient’s life. Despite major improvements in treatment and care of ARM patients in the past decade, a substantial number of patients face lifelong physical and psychosocial problems[4].

This study aimed to identify rare genetic variants associated with ARM by analyzing exome chip data of >240,000 known rare and low-frequency coding variants in a large ethnically homogeneous series of 568 Caucasian ARM patients and 1,860 Caucasian population-based controls. We identified 13 variants that were statistically significantly associated with ARM, but all of these appeared to be false-positive findings. Therefore, this first exome chip study did not provide statistical evidence for association of rare genetic variants with large effect sizes and ARM.




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