A maternally inherited chromosome 18q22.1 deletion in a male with late-presenting diaphragmatic hernia and microphthalmia-evaluation of DSEL as a candidate gene for the diaphragmatic defect

Abstract

Using an Affymetrix GeneChip(R) Human Mapping 100K Set to study a patient with a late-presenting, right-sided diaphragmatic hernia and microphthalmia, we found a maternally inherited deletion that was 2.7 Mb in size at chromosome 18q22.1. Mapping of this deletion using fluorescence in situ hybridization revealed three deleted genes-CDH19, DSEL, and TXNDC10, and one gene that contained the deletion breakpoint, CCDC102B. We selected DSEL for further study in 125 patients with diaphragmatic hernias, as it is involved in the synthesis of decorin, a protein that is required for normal collagen formation and that is upregulated during myogenesis. We found p.Met14Ile in an unrelated patient with a late-presenting, anterior diaphragmatic hernia. In the murine diaphragm, Dsel was only weakly expressed at the time of diaphragm closure and its expression in C2C12 myoblast cells did not change significantly during myoblast differentiation, thus reducing the likelihood that the gene is involved in myogenesis of the diaphragm. Although it is possible that the 18q22.1 deletion and haploinsufficiency for DSEL contributed to the diaphragmatic defect in the patient, a definite role for DSEL and decorin in the formation of the collagen-containing, central tendon of the diaphragm has not yet been established.

Fig. 1

Array Studies Showed that the Propositus has a 2.7 Mb Deletion at 18q22.1. Chromosome 18, Log₂Ratio from an Affymetrix 100K Array in the propositus showing a 2.7 Mb deletion at Chromosome 18q22.1. A chromatogram showing the G-banding pattern for chromsome 18 is located under the figure. The vertical bars of the figure show: GSA_CN (Genome smoothed average, copy number), GSA_pVal (Genome smoother average, p value), log₂ratio and LOH (loss of heterozygosity).

Fig. 2

Re-sequencing of DSEL Reveals a Novel Missense Substitution, p.Met14Ile, in an Unrelated Patient with Diaphragmatic Hernia. Chromatogram showing c.42G>A (indicated by arrow) in forward and reverse chromatograms, predicting p.Met14Ile in DSEL.

Fig. 3

In-situ Hybridization of the Developing Diaphragm Showed Weak Expression of Dsel at the time of Diaphragm Closure. In-situ hybridization using antisense and sense riboprobes for Dsel, showing weak expression in the murine diaphragm at E13.5.

Fig. 4

RT-PCR Showed no Significant Change in Dsel Expression During the Differentiation of C2C12 cells. Graph showing mean and standard deviation of Dsel and Hprt Ct at day 0, 3 and 6 of C2C12 cell differentiation, showing minimal change of Dsel expression with C2C12 cell differentiation between day 0 (undifferentiated cells) and day 6 (fully differentiated cells). Each experimental point was repeated in quadruplicate reactions