Genomic assessments of the frequent loss of heterozygosity region on 8p21.3-p22 in head and neck squamous cell carcinoma

Abstract

Most human cancers are characterized by genetic instabilities. Chromosomal aberrations include segments of allelic imbalance identifiable by loss of heterozygosity (LOH) at polymorphic loci, which may be used to implicate regions harboring tumor suppressor genes. Here we performed whole-genome LOH profiling on 41 human head and neck squamous cell carcinoma (HNSCC) cell lines. Several frequent LOH regions were identified on chromosomal arms 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11q, and 17p. A genomic region of approximately 7 Mb located at 8p21.3 approximately p22 exhibits the most frequent LOH (87.9%), which suggests that this region harbors one or more important tumor suppressor genes. Mitochondrial tumor suppressor gene 1 (MTUS1) is a recently identified candidate tumor suppressor gene that resides in this region. Consistent downregulation in expression was observed in HNSCC for MTUS1 as measured by real-time quantitative reverse transcriptase-polymerase chain reaction. Sequence analysis of MTUS1 gene in HNSCC revealed several important sequence variants in the exon regions of this gene. Thus, our results suggest that MTUS1 is one of the candidate tumor suppressor genes for HNSCC residing at 8p21.3 approximately p22. The identification of these candidate genes will facilitate the understanding of tumorigenesis of HNSCC.

Figure 1

SNP array based LOH profiles of chromosome 8 in HNSCC The genomic DNA from each cell lines was assayed using Affymetrix 10K SNP mapping array. The LOH regions were detected and demarcated using dChipSNP as described [18]. Each column represents one cell line, and each row represents a SNP marker. Color code: Blue = LOH; Yellow = retained; Gray = uninformative; White = no call. The relative genomic location was indicated by the cytoband map on the left. The blue curve in the shaded gray box on the right denotes LOD (Logarithm of the odds) score representing the excessive sharing of LOH. The red arrow head designates the most frequent LOH region at chromosome 8p. Loss of heterozygosity (LOH) map: single-nucleotide polymorphism (SNP) array-based LOH profiles of chromosome 8 in head and neck squamous cell carcinoma (HNSCC). The genomic DNA from each cell lines was assayed using an Affymetrix 10K SNP mapping array. The LOH regions were detected and demarcated using dChipSNP informatics software, as previously described [18]. Each column represents one cell line, and each row presents a SNP marker, with colours as follows: blue, LOH; yellow, retained; gray, uninformative; white, no call. The relative genomic location is indicated by the cytoband map (left). In the shaded gray box (right), the blue curve denotes the logarithm of the odds score (LOD score) representing the excessive sharing of LOH. The red arrowhead designates the most frequent LOH region at chromosome 8p.

Figure 2

Genes located in the frequent LOH region at 8p22−21.3 The frequent LOH region at 8p22−21.3 was displayed using UCSC Genome Browser. Genes located in this genomic region and their corresponding mRNAs were plotted. Genes located in the frequent LOH region at 8p21.3∼p22, as displayed using the UCSC Genome Browser (http://www.genome.ucsc.edu). Genes located in this genomic region and their corresponding mRNAs were plotted.

Figure 3

The mRNA level of MTUS1 in HNSCC. The qRT-PCR for MTUS1 was performed as described in Methods and Materials. The data analysis was carried out using the 2^{-delta delta Ct} method described previously [22], where beta-actin was used as reference gene. A) The mRNA levels of MTUS1 were evaluated on 10 HNSCC cell lines and normal oral keratinocytes (NHOK). B) The mRNA levels of MTUS1 were evaluated on 10 pairs of tongue SCC cases and matching normal tissue samples. The mRNA level of MTSU1 in HNSCC. Quantitative reverse transcriptase–polymerase chain reaction for MTUS1 was performed as described in Methods and Materials. Data analysis was performed using the 2^–ΔΔCt method as described previously [22], with the β-actin gene ACTB used as the reference. (A) The mRNA levels of MTUS1 were evaluated on 10 HNSCC cell lines and normal oral keratinocytes (NHOK). (B) The mRNA levels of MTUS1 were evaluated on 10 pairs of oral tongue squamous cell carcinoma cases (OSCC) and matching normal tissue samples.

Figure 4

Exonic sequence variations of MTUS1 gene in HNSCC. The schematic representation of the genomic organization of MTUS1 gene located on the minus strand of chromosome 8p22 was presented. The genomic locations of the detected nucleotide sequence variants for MTUS1 gene in HNSCC were indicated. The nucleotide sequence variants that lead to amino acid changes were identified with bold font. The * indicates the nucleotide sequence variants were identified previously [23]. Exonic sequence variations of MTUS1 gene in HNSCC: a schematic representation of the genomic organization of MTUS1 gene located on the minus strand of chromosome 8p22. The genomic locations of the detected nucleotide sequence variants for MTUS1 gene in HNSCC are indicated. Boldface type identifies the nucleotide sequence variants that lead to amino acid changes. An asterisk marks the nucleotide sequence variants identified previously [23].

Figure 5

Sequence analyses of the MTUS1 gene in HNSCC. Sequence analyses of MTUS1 gene were performed on 10 HNSCC cell lines as described. A total of 13 single nucleotide sequence variants were observed in exon 1, 2, 8, 9, 11, and 17. The representative sequence chromatograms for nucleotide sequence variants varA (Cys-Arg), varB (Thr-Ser), varE (Lys-Thr), and varL (Leu-Val) were presented. The arrows designate nucleotide variants. Sequence analyses of the MTUS1 gene were performed on HNSCC cell lines as described. A total of 13 single-nucleotide sequence variants were observed, in exons 1, 2, 8, 9, 11, and 17. Shown here are representative sequence chromatograms for nucleotide sequence variants varA (Cys-Arg), varB (Thr-Ser), varE (Lys-Thr), and varL (Leu-Val). Arrows indicate nucleotide variants.