Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH

Abstract

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N=179) for microdeletions in this interval by the multiplex ligation-dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0-like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left-sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patient's lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene.

Figure 1

MLPA results for 1q41q42.12 microdeletion screening. Normalized peak height graphs are shown for two CDH patients. (A) a CDH patient without a 1q41q42.12 microdeletion (B) Patient 3, the previously reported Fryns phenotype patient [Kantarci et al., 2006]. Each bar represents the normalized peak height for the probe focusing on a gene on the X-axis, and is arranged in genomic order. Bars 1-2: Contol regions on 15q26. Bar 3: PTPN14 (212,768,242-212,791,265 at 1q41); Bar 4: USH2A (214,413,915-214,663,361 at 1q41); Bar 5 EPRS (218,227,107-218,286,623 at 1q41); Bar 6: BPNT1 (218,297,696-218,329,814 at 1q41); Bar 7: DUSP10 (219,941,389-219,982,084 at 1q41); Bar 8: CAPN2 (222,011,949-222,030,343 at 1q41); Bar 9: LBR (223,660,887-223,682,407at 1q41.12); Bar 10: ACBD3 (224,399,003-224,441,046 at 1q41.12); Bar 11: ITKPKB (224,961,518-224,993,499 at 1q41.12); Bar 12: PSEN2 (225,129,689-225,150,427 at 1q41.12 at 1q41.13); and Bar 13: TAF5L (227,801,564-227,828,417 at 1q42.13). Note that the deletion extends from EPRS to ACBD3 in (B) Patient 3.

Figure 2

The family pedigree and sequencing chromatograms of the DISP1-exon 8 showing a de novo heterozygous mutation [c.4412C>G (p.Ala1471Gly)] on lymphoblastoid cell line derived DNA of Patient 1. The peripheral blood derived DNA of the patient demonstrates a low level of the mutant allele, while there is no detectable mutant allele in his saliva derived DNA, indicative of tissue specific mosaicism. Arrows show the heterozygous C to G substitution in the patient.

Figure 3

Restriction enzyme digestion of a 195-bp DISP1 exon 8 product with CviKI-1. Wild type product with CviKI-1 recognition site (GG ↓CT) is digested into fragments of 103-bp and 92-bp. The mutation [c.4412C>G (p.Ala1471Gly)] abolishes a CviKI-1 site (GG GT). The presence of the fragments of 195-bp, 103-bp and 92-bp indicates a heterozygous mutation. Samples are as follows: Promega 100-bp DNA ladder (M); undigested PCR product (lane 1); Patient 1’s lymphoblastoid cell line (lane 4), peripheral blood (lane 6), and saliva (lane 9); mother’s lymphoblastoid cell line (lane 2) and saliva (lane 7); father’s lymphoblastoid cell line (lane 3) and saliva (lane 8); healthy brother’s saliva DNA (lane 10); and wild type control (lane 5). Both lane 4 (lymphoblastoid cell line DNA) and lane 6 (peripheral blood DNA) show a heterozygous mutation in Patient 1. Note that the 195-bp mutant fragment is less intense in lane 6 compared to lane 4. There is no observed mutant fragment in the patient’s saliva DNA (lane 9).

Figure 4

Pyrosequencing analysis of the DISP1 mosaic mutation [c.4412C>G (p.Ala1471Gly)] in Patient 1 and his family members. The mutated base (C>G) is highlighted in yellow. Quantitative analysis identifies that the mutation is present in 43%, 12%, 4.5% of the patient’s samples from lymphoblastoid cell line, peripheral blood cells, and saliva, respectively.

Figure 5

A) Schematic representation of DISP1 protein and the position of p.Ala1471Gly mutation. B) The protein sequence alignment by ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) in different species shows that wild type alanine (A) at position 1471 is evolutionarily conserved. Conserved amino acids compared to humans are indicated in red.

Figure 6

Disp1 and Hedgehog pathway genes in the diaphragm. In situ hybridization on E11.5 mouse embryos (A-I) and X-gal staining (J at E11.5 and K-M mature diaphram) during diaphragm development. Developing diaphragmatic tissue including PPF is outlined with dotted lines in A-J. Disp1 has a generally diffuse pattern of expression but is not expressed in the heart (shown in sections A-D as negative control). Disp1 is highly expressed in the pleuroperitoneal folds (PPFs), especially in the region adjacent to the inferior vena cava (C, D). The expression pattern in the lung includes cells from the mesothelium, mesenchyme, and epithelium (A-F). Indian hedgehog (Ihh) is not expressed in the PPF but is highly expressed in the stomach (G). Patched1 is expressed in the esophagus, lung, and stomach but not in the diaphragm (H). Shh is expressed in lung epithelium but not in the PPF (I). Shh expressing cells (Shh ^GFP-Cre) do not contribute to the developing diaphragm tissue but do contribute to the lung and esophagus (J). X-gal staining of mature diaphragms from a Shh-Cre fate mapping experiment show that Shh expressing cells contribute to the esophagus (red arrow) and phrenic nerve (black arrows) but not to the mature diaphragmatic parenchyma (K, L). A control littermate (stained but not Cre expressing) shows no staining of the lung, diaphragm, or esophagus (M). The insert shows a piece of lung as a positive control (M, inset). (lu-lung, ht-heart, IVC-inferior vena cava, li-liver, st-stomach)