November 9, Johns Hopkins School of Medicine, Baltimore, MD,

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1 Fast detection of de-novo copy number variants from case-parent SNP arrays identifies a deletion on chromosome 7p14.1 associated with non-syndromic isolated cleft lip/palate Samuel G. Younkin 1, Robert B. Scharpf 2, Mary L. Marazita 3, Alan F. Scott 2,TerriH.Beaty 1, Ingo Ruczinski 1 November 9, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 2 Johns Hopkins School of Medicine, Baltimore, MD, 3 University of Pittsburgh, School of Dental Medicine, Pittsburgh, PA. Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 1 Background Copy number variants (CNVs) may play an important part in the development of common birth defects such as oral clefts, and individuals with multiple birth defects (including clefts) have been shown to carry small and large chromosomal deletions. Here we investigate de novo deletions, i. e. segments of the genome in which a deletion is present in a child with cleft lip/palate, but absent in both healthy parents. We compare de novo deletion frequencies in the oral cleft trios to those in trios with children not selected for a particular phenotype. Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 2

2 Methods We collected case-parent trios as part of the Oral Cleft Project in the GENEVA Consortium, and were generously provided with trio data from small pedigrees collected from rural Appalachia as part of a study of dental caries performed by Marazita et al. to serve as controls. We used two methods to identify de novo deletions the joint hidden Markov model (HMM) implemented in PennCNV, and a novel approach named MinimumDistance that first segments the parent to offspring differences in marker probe intensity, and then uses maximum a posteriori estimation to infer de novo CNVs from the segmented genome. Both methods rely on measures of the marker probe intensity and B allele frequency generated by hybridization to the Illumina 610 quad array as performed by the Center for Inherited Disease Research (CIDR) at Johns Hopkins University. Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 3 Minimum Distance (1) Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 4

3 Minimum Distance (2) In the second step maximum a posteriori estimation can for example discriminate between de-novo hemizygous deletions (left) and inherited homozygous deletions (right). Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 5 Quality control All parent-offspring trios were of Caucasian ethnicity. Only de novo deletions that spanned at least ten markers were included. The frequency of de novo deletions was greatly affected by DNA source and study protocol. Trios with at least one whole-genome amplified sample and/or LRR median absolute deviation above 0.3 were excluded. The usual PennCNV quality control procedures were applied. Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 6

4 Methods / Results We decomposed the set of all de novo deletions into CNV components, i. e. partitions containing no partially overlapping CNVs. For each of these regions with at least 5 de novo deletions in the cleft group and control group combined we performed a one-sided Fisher s exact test. To correct for the correlation among adjacent CNV components and to take multiple tests over all CNV components into account, we derived genome-wide significance at a 5% family-wide error rate through permutations. Among the 321 (455) de novo deletions found with MinimumDistance (PennCNV ) is a genome-wide significant peak on chromosome 7p14.1. [Figurenextslide] Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 7 Results log 10 p MD PennCNV Chromosome Manhattan plot of the log 10 p-values for MinimumDistance (blue) and PennCNV (red). The horizontal dashed lines indicates permutation-based genome-wide significance. Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 8

5 Results The most significant association based on MinimumDistance was observed in a region with 13 de novo deletions among the 470 cleft lip/palate probands, and no de novo deletions in the 391 control trios. The most significant PennCNV association was observed in a region with 21 de novo deletions among the probands, and one de novo deletion in the controls. Count TARP cleft (Penn) cleft (MD) control (Penn) control (MD) Chromsome 7 (MB) Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 9 Results In the above 80 kb region on chromosome 7 we observed 22 oral cleft subjects with a de novo deletion, while no more than two individuals in the control trios carry such a deletion. Of these 22 cleft lip/palate subjects with a de novo deletion nine had cleft lip, seven had cleft palate and six had cleft lip and palate. The nearest gene to this region is the TARP gene which has not been associated previously with facial abnormalities. Other genes in the vicinity include AMPH, FAM183B, STARD3NL and TXNDC3. http: //biostat.jhsph.edu/ iruczins/ ingo@jhu.edu Samuel G. Younkin et al (JHU) Fast detection of de-novo copy number variants in case-parent trios 10