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. 2009 May;75(5):429-39.
doi: 10.1111/j.1399-0004.2009.01182.x.

Sequence variants in the HLX gene at chromosome 1q41-1q42 in patients with diaphragmatic hernia

A M Slavotinek  1 , A Moshrefi, N Lopez Jiminez, R Chao, A Mendell, G M Shaw, L A Pennacchio, M D Bates
Affiliations

Sequence variants in the HLX gene at chromosome 1q41-1q42 in patients with diaphragmatic hernia

A M Slavotinek et al. Clin Genet. 2009 May.

Abstract

Congenital diaphragmatic hernia (CDH) is a common birth defect for which few causative genes have been identified. Several candidate regions containing genes necessary for normal diaphragm development have been identified, including a 4-5 Mb deleted region at chromosome 1q41-1q42 from which the causative gene(s) has/have not been cloned. We selected the HLX gene from this interval as a candidate gene for CDH, as the Hlx homozygous null mouse has been reported to have diaphragmatic defects and the gene was described as being expressed in the murine diaphragm. We re-sequenced HLX in 119 CDH patients and identified four novel single nucleotide substitutions that predict amino acid changes: p.S12F, p.S18L, p.D173Y and p.A235V. These sequence alterations were all present in patients with isolated CDH, although patients with both isolated CHD and CDH with additional anomalies were studied. The single-nucleotide substitutions were absent in more than 186 control chromosomes. In-situ hybridization studies confirmed expression of Hlx in the developing murine diaphragm at the site of the junction of the diaphragm and the liver. Although functional studies to determine if these novel sequence variants altered the inductive activity of Hlx on the alpha-smooth muscle actin and SM22alpha promoters showed no significant differences between the variants and wild-type Hlx, sequence variants in HLX may still be relevant in the pathogenesis of CDH in combination with additional genetic and environmental factors.

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Figures

Fig. 1
Fig. 1
(a) Chromatogram showing c.35C>T, predicting p.S12F in the HLX gene from a patient with a right-sided congenital diaphragmatic hernia. (b) Chromatogram showing c.53C>T, predicting p.S18L in the HLX gene from a patient with a right-sided congenital diaphragmatic hernia. (c) Chromatogram showing c.517G>T, predicting p.D173Y in the HLX gene from a patient with a right-sided congenital diaphragmatic hernia. (d) Chromatogram showing c.704C>T, predicting p.A235V in the HLX gene from a patient with a left-sided congenital diaphragmatic hernia.
Fig. 2
Fig. 2
Alignment of the HLX gene showing conserved residues at the site of the sequence variants predicting p.S12F, p.S18L, p.D173Y and p.A235V. The top bar shows conservation among the 17 species shown in the figure. Red indicates that the amino acids are identical in all 17 species; orange indicates that the amino acids are identical in 14–16 species; green indicates that the amino acids are identical in 11–12 species; light blue indicates that the amino acids are identical in 7–10 species; dark blue indicates that the amino acids are identical in four to six species; black indicates that the amino acids are identical in three species and white indicates that the amino acids are identical in one to two species. The second bar shows conservation among the eight mammalian species. Red indicates that the amino acids are identical in all eight species; orange indicates that the amino acids are identical in seven species; green indicates that the amino acids are identical in five to six species; light blue indicates that the amino acids are identical in four species; dark blue indicates that the amino acids are identical in three species.
Fig. 3
Fig. 3
(a) In-situ hybridization studies using a full-length Hlx antisense probe (Fig. 3a) and an Hlx sense probe at E11.5 (first panel), E12.5 (second panel), E13.5 (third panel) and E14.5 (fourth panel). In the E11.5 antisense panel: He, heart; hp, hepatic primordium. The developing diaphragm is located between these two structures (37). In the E12.5, E13.5 and E14.5 antisense panels, the arrows indicate the developing diaphragm. Expression of Hlx can be seen in the murine diaphragm and adjacent liver capsule with the antisense probe but not the sense probe, and expression appears strongest at E13.5. Hlx is also strongly expressed in the developing liver. (Fig. 3b) shows a higher magnification view (40×) from the E13.5 antisense section, site indicated by the box in Fig. 3a. Diaphragm staining is indicated by an arrow.
Fig. 4
Fig. 4
Functional studies of the ability of wild-type Hlx and Hlx constructs with sequence variants to induce α-smooth muscle actin and SM22α promoter activity in a cell line derived from an Hlx−/− mouse. (a) The results for the α-smooth muscle actin promoter are shown. The x-axis has bars representing the different sequence variants and the y-axis has the values for ratios of firefly luciferase (expressed by pGL3 constructs with promoters of interest, expressed in relative luciferase units, or RLUs) to Renilla luciferase (expressed by pRL-SV40) for triplicate wells in duplicate experiments. (b) The results for the SM22α promoter are shown. The x-axis has bars representing the different sequence variants and the y-axis has the values for ratios of firefly luciferase (expressed by pGL3 constructs with promoters of interest, expressed in RLUs) to Renilla luciferase (expressed by pRL-SV40) for triplicate wells in duplicate experiments.
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