HLX1 gene variants influence the development of childhood asthma.

BACKGROUND: Major transcription factors controlling T(H)1 and T(H)2 development, such as T-box transcription factor and GATA3, might be centrally involved in asthma and atopic diseases. Only recently, the homeobox transcription factor H.20-like homeobox 1 (HLX1), interacting closely with T-box transcription factor, has been identified as an important regulator of T(H)1 differentiation and suppressor of T(H)2 commitment. OBJECTIVE: We investigated whether genetic variations in the HLX1 gene exist and whether these could affect the development of childhood asthma. METHODS: The HLX1 gene was resequenced in 80 chromosomes. Associations between identified polymorphisms, asthma, and atopic diseases were investigated in German children (total n = 3099) from the cross-sectional International Study of Asthma and Allergy in Childhood phase II. Functional properties of polymorphisms were studied by using luciferase reporter gene assays and electrophoretic mobility shift assays in T cells. All statistical analyses were performed with SAS/Genetics software (SAS Institute, Inc, Cary, NC). RESULTS: Nineteen polymorphisms were identified in the HLX1 gene, and 2 tagging single nucleotide polymorphisms representing 7 polymorphisms were associated with childhood asthma in our study population. Two promoter polymorphisms, C-1407T and C-742G, contained in 1 tagging block were associated with asthma (odds ratio, 1.44; 95% CI, 1.11-1.86; P = .0061), significantly decrease promoter transactivation, and disrupt specificity protein-transcription factor binding in in vitro experiments. CONCLUSIONS: Our data suggest that polymorphisms in the HLX1 gene increase the risk for childhood asthma. On the cellular level, altered binding of specificity protein-transcription factors to the HLX1 promoter and subsequent changes in HLX1 gene expression might contribute to these effects.

Molecular analysis of TGF-(beta)s and their receptors in human keratinocyte cell lines of different biological behaviour.

We investigated three clonally related human keratinocyte cell lines of different biological behaviour, HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic), with regard to the expression of TGF-beta-isoforms -1, -2 and -3 and that of the TGF-beta-cell-receptors TBR-I, -II and -III. In addition, we amplified and sequenced the genome of TBR-II which is known to be a target for mutations in several types of malignant tumors including squamous cell carcinomas. In all three cell lines, TGF-beta1 and -beta3 were present only in very low amounts. Western blots provided no evidence for differences in TGF-beta1 between the cell lines. However, in immunohistochemistry more cells were slightly positive for this cytokine in HaCaT than in A5 and II-4RT cells. In contrast, a significantly variable expression of TGF-beta2 was seen by both Western blot and immunohistochemistry. Thereby, the non-tumorigenic HaCaT-cells contained significantly more TGF-beta2 than the tumorigenic, benign A5 cells and the malignant II-4RT cells. TBR-I, -II and -III were present in all three cell lines. While most cells were positive for TBR-I, only part of the cells contained TBR-II and -III, however, without obvious differences between the three cell lines. The molecular analysis of all 7 exons of TBR-II by PCR amplification and direct sequencing revealed in all three cell lines correct sequences without evidence for mutations. Our study indicates differences in the expression of TGF-beta in a human model of keratinocytes of varying tumorigenicity, but presents no evidence for mutations in the functionally most important TGF-beta-receptor TBR-II. This suggests a dysregulation of cytokine control on the level of TGF-beta expression, which may be responsible for the biological behaviour.

High frequency of TGF-beta-receptor-II mutations in microdissected tissue samples from laryngeal squamous cell carcinomas.

In this study we analyze 105 paraformaldehyde-fixed and paraffin-embedded tumor samples from 12 patients with invasive squamous cell carcinoma of the larynx for the presence of gene mutations of the complete TGF-beta-receptor-II (TBR-II) gene. This study was conducted on tissue samples following separation of tumor cell groups from adjacent stroma cell compartments by laser microdissection, resulting in pure tumor cell complexes of approximately 50 to 500 cells. We detected 35 different mutations in 5 of the 12 patients analyzed but none in numerous samples of the normal peritumoral stroma or in normal epithelium. Twelve of the mutations were silent and nonfunctional, whereas the 23 relevant mutations were either bp replacements leading to amino acid exchanges or deletions leading to frame shifts and premature stop codons. Except for the so-called "big polyadenine tract" in exon 3 with several similar mutations, no further mutational hot spot was found. In addition we found a correlation between mutations and a loss of typical TGF-beta effects in tumor cells (high cell proliferation rate) but not in the stroma cells (low proliferative capacity, significant de novo deposition of matrix material). This study is the first to identify a high mutational rate of the TBR-II gene in laryngeal squamous cell carcinoma. We show that that only small tumor-cell groups are affected. The molecular abnormalities are variable, and only one hot spot of mutations can be identified (exon 3, big polyadenine tract). These defects and possibly comparable mutations in other proteins of the TGF-beta-signaling cascade seem to be associated with enhanced cell proliferation rates and alterations of the peritumoral matrix.