Table 1.
Overview of the phenotypic characteristics of the patient group.
Figure 1.
CNV discovery and characterization.
Molecular karyotyping was performed for 200 patients with short stature after thorough clinical evaluation and for 820 healthy control samples. Exclusion of common variants using the control samples and scoring CNVs above 50 kb resulted in an approx. reduction of 80% of the CNVs identified in the patient samples. 60.5% of these CNVs affect reference sequence gene regions. Functional characterization includes segregation analysis using parental arrays and/or MLPA as well as gene and CNV based evaluation.
Figure 2.
Molecular karyotyping and MLPA confirmation of identified loci.
(A) Example representation of the copy number analysis of patient 1 using the Affymetrix Genotyping Console 3.0.2 software. The red bar shows the 2.2 Mb deletion region (CNSegments). (B) Graphical presentation of the deletion region including 33 candidate genes (modified from UCSC genome browser). (C) MLPA confirmation with a probe in the NFASC gene region. A relative quantity value (RQ) below 0.75 was considered as confirmation of a deletion, above 1.25 as confirmation of a duplication. Detailed data for the remaining patients is presented in the supporting information.
Figure 3.
Higher incidence of CNVs with a length of above 100 kb in affected individuals.
(A) Presentation of the Odds Ratio (light blue) and –log10(p-value) (dark blue) for determination of the size threshold of the number of CNVs in patients vs. controls. The Odds Ratio and the –log10(p-value) confirms a CNV size cut-off at 99.2 kb (OR 1.26 and p-value 4.98×10−8). (B) Fraction of copy numbers segments in cases (grey) vs. control (white) quintiles (* p<0.005). Quintile borders were Q1: 68.3 kb, Q2: 99.3 kb, Q3 149.6 kb, Q4 298.1 kb, and Q5: 72,571.3 kb. The y axis presents the fraction of CNVs inside the corresponding quintile bin. Significance levels are calculated using Fisher's exact test. The figure shows a shift towards segments above 100 kb in patients (** p-value 1.188×10−7).
Table 2.
Lines of Evidence.
Figure 4.
Height distribution of the study group.
Height distribution (SDS) of all 200 patients (blue) compared to the 20 patients with identified CNVs (green). The mean SD score for height was −3.34. Patients with identified CNVs showed a significant difference in the SDS distribution (p-value 0.03).
Table 3.
Summary phenotype of patients with identified CNVs.
Figure 5.
Genome-wide significant association of GWAS loci for height distribution in the 3 CNVs.
The values of r2 are based on the CEU 1000genomes Nov 2010 samples. The blue line and right-hand y axis represent recombination rates. The SNPs with the min p values are highlighted as purple diamond. The figures were created using LocusZoom (http://csg.sph.umich.edu/locuszoom/).