High Mobility Group Protein Gene Research Articles

Evaluation of the reliability of maize reference assays for GMO quantification

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Cell‐free DNA in amniotic fluid remains to be attached to HMGA2–implications for noninvasive prenatal diagnosis

The expression of the high mobility group protein gene HMGA2 is primarily confined to embryonic and fetal cells. The aim of this study was to determine the relative expression level of HMGA2 in cells of amniotic fluid samples. Furthermore, it should be investigated by chromatin immunoprecipitation whether or not HMGA2 is attached to cell-free DNA in amniotic fluid. Expression levels of HMGA2 in 58 amniotic fluid samples from the second trimester were measured using quantitative real-time polymerase chain reaction (PCR). Furthermore, the presence of HMGA2, attached to cell-free DNA was tested by chromatin immunoprecipitation. Expression of HMGA2 was detected in all samples, but in cells of the amniotic fluid it was 161-fold higher than in cells of the urine from healthy donors. The real-time PCR with GAPDH showed a signal in all samples treated with the improved protocol of immunoprecipitation. Our data clearly show that cells of the amniotic fluid strongly overexpress HMGA2 according to their fetal origin. The fact that apparently HMGA2 remains to be attached to cell-free DNA suggests interesting new approaches in noninvasive prenatal diagnosis.

Interlaboratory Trial Validation of an Event-Specific Qualitative Polymerase Chain Reaction-Based Detection Method for Genetically Modified RT73 Rapeseed

The qualitative event-specific polymerase chain reaction detection method of genetically modified (GM) RT73 rapeseed was developed based on the cloned 3' end flanking sequence of RT73 rapeseed integration. The specificity of the method for GM RT73 rapeseed was validated using several different GM rapeseed lines, GM maize lines, GM soybean line, non-GM rapeseed, and other non-GM crops. In this study, the developed method was validated through an interlaboratory study by 12 laboratories from 6 countries. The sensitivity of this method was evaluated using several mixed rapeseed meals with different GM RT73 rapeseed contents from 5.0 to 0.01% prepared by our laboratory. The evaluated results showed that all of the rapeseed endogenous reference high mobility group protein gene (HMG I/Y), figwort mosaic virus 35S (FMV 35S) promoter, and RT73 event-specific fragment could be detected from rapeseed samples at 0.1% (w/w) with a confidence level of more than 95%. All results from the 12 laboratories indicated that the developed method could be considered fit for the detection and identification of GM RT73 rapeseed.

Upregulation of HMGA2 in thyroid carcinomas: A novel molecular marker to distinguish between benign and malignant follicular neoplasias

The identification of molecular markers allowing to differentiate between benign and malignant thyroid tumors remains a diagnostic challenge. Herein, we have used the expression of the high mobility group protein gene HMGA2 and its protein, respectively, as a possible marker detecting malignant growth of thyroid tumors. HMGA2 belongs to the high mobility group proteins, i.e. small, highly charged DNA-binding proteins. While HMGA2 is highly expressed in most embryonic tissues, its expression in adult tissues is very low. However, a reactivation of HMGA2 expression has been described for various malignant tumors and often correlates with the aggressiveness of the tumors. The aim of this study was to investigate whether the HMGA2 expression can be used to detect malignant thyroid tumors. RNA from 64 formalin-fixed paraffin-embedded thyroid tissues including normal tissue (n = 3), thyroiditis (n = 2), and follicular adenomas (n = 19) as well as follicular (n = 9), papillary (n = 28), and anaplastic (n = 3) carcinomas was reverse transcribed. Finally, real-time quantitative RT-PCR was performed. Expression differences of up to 400-fold were detected between benign and malignant thyroid tumors. Based on HMGA2 expression alone, it was possible to distinguish between benign and malignant thyroid tissues with a sensitivity of 95.9% and a specificity of 93.9%. There was a highly significant (P < 0.001) difference with histology of the tumors being the gold standard between the benign lesions and malignant tumors. Our results show that even as a stand-alone marker HMGA2 expression has a high potential to improve diagnoses of follicular neoplasms of the thyroid.

Localization of gender-regulated gene expression in the filarial nematode Brugia malayi

We used in situ hybridization (ISH) to localize expression of gender-biased genes in the filarial parasite Brugia malayi that were previously identified by microarray analysis and quantitative reverse transcriptase PCR (qRT-PCR). We studied seven genes with male-biased expression, 11 genes with female-biased expression, and one control gene with equal expression in males and females. RNA probes were hybridized to frozen sections of adult worms. ISH confirmed gender-biased expression for all 18 of the differentially expressed genes and non-biased expression for the control. We identified six patterns of expression for these genes. As expected, most of the gender-biased genes were expressed in reproductive organs, developing gametes and embryos. Hybridization signal intensities correlated with relative mRNA levels as assessed by qRT-PCR. Some of the differentially expressed genes had tightly regulated expression patterns. For example, a high mobility group protein gene ( Bm-hmg) was exclusively expressed in developing larvae in females. Expression was first detected in late stage oocytes, peaked in morula stage embryos and no signal was detected in late pretzel stage or in stretched microfilariae. Another female up-regulated gene (microfilarial sheath protein Bm- shp-1) was exclusively expressed in the epithelium of uterine sections that contained morulae or early pretzel embryos. No signal was detected in other female structures, in late embryos or in male worms. This result suggests that microfilarial sheath proteins are produced by the uterus epithelium and not by embryos. Transcripts of the male-upregulated major sperm protein-1 ( Bm-msp-1) were detected in spermatocytes in the early spermatogenesis zone and in spermatids but not in spermatozoa in the vas deferens. Thus, ISH provides a means to independently confirm differential expression of genes identified by other methods. In addition, localization patterns provide insight regarding the function of known or novel genes in the parasite.

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52–54°C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Absence of HMGIC-LHFP fusion in pulmonary chondroid hamartomas with aberrations involving chromosomal regions 12q13∼q15 and 13q12∼q14

In a variety of benign solid human tumors the high mobility group protein gene HMGIC is affected by aberrations involving the chromosomal region 12q14∼q15. Beside the two predominant alterations t(3;12) (q27∼28;q14∼q15) and t(12;14)(q14∼q15;q23∼q24), the t(12;13)(q14∼q15;q12∼q14) is another aberration observed recurrently in these tumors. Very recently, an HMGIC-LHFP ( lipoma HMGIC fusion partner) fusion gene has been detected in a lipoma with a t(12;13). The results of the present study demonstrated the absence of the HMGIC-LHFP fusion in three pulmonary chondroid hamartomas (PCH) with complex aberrations involving chromosomal regions 12q13∼q15 and 13q12q∼q14 and one PCH with a simple t(12;13)(q14∼15;q13) by reverse transcription–polymerase chain reaction. Thus, intragenic rearrangements within the LHFP gene leading to its fusion to HMGIC are not a consistent finding in mesenchymal tumors with clonal aberrations of both chromosomal regions 12q13∼q15 and 13q12∼q14.

Large deletion of part of the HMGIC locus accompanying a t(3;12)(q27∼q28;q14∼q15) in a lipoma

The high mobility group protein gene HMGIC has been found to be rearranged in a variety of human benign solid tumors. Often, these rearrangements lead to fusion genes of which that between HMGIC and LPP underlying t(3;12)(q27∼q28;q14∼q15) is by far the most common. Herein, we analysed a lipoma with a t(3;12)(q27∼q28;q14∼q15). RT-PCR revealed the presence of a HMGIC-LPP fusion transcript composed of exons 1–3 of HMGIC and exons 9–11 of LPP, but the absence of the reverse LPP-HMGIC fusion transcript. Fluorescence in situ hybridization experiments using different probes derived from the HMGIC gene and its 3′ vicinity showed the absence of FISH signals on the derivative chromosome 3. Thus, in the present tumor the t(3;12)(q27∼q28;q14∼q15) was accompanied by a large genomic deletion. Roughly, it can be estimated that at least 170 kb of chromosome 12 material were deleted. To the best of our knowledge, this is the first report showing that the “simple” t(3;12)(q27∼q28;q14∼q15) results in a large deletion of DNA.

A novel LPP fusion gene indicates the crucial role of truncated LPP proteins in lipomas and pulmonary chondroid hamartomas

The lipoma preferred partner (LPP) gene is fused to the high mobility group protein gene HMGIC in lipomas and pulmonary chondroid hamartomas. In addition, a fusion of LPP to the mixed lineage leukemia (MLL) gene has been described in a case of acute myeloid leukemia. Herein, a novel LPP fusion transcript of LPP in a lipoma is described that points to the possible oncogenic potential of LPP.

Evidence for RAD51L1/HMGIC fusion in the pathogenesis of uterine leiomyoma.

Chromosome rearrangements involving 12q15 are frequently observed in a variety of human mesenchymal tumors. The high mobility group protein gene HMGIC has been identified as the target involved in these rearrangements. Uterine leiomyomas frequently use chromosome band 14q24 as a translocation partner to HMGIC. Recent studies within the chromosome 14 breakpoint region in cell lines carrying t(12;14)(q15;q23-24) revealed that RAD51L1, a member of the RAD51 recombination gene family, is the HMGIC partner. Using RT-PCR, we screened a panel of 81 uterine leiomyomas removed from 30 women for rearrangement between RAD51L1 and HMGIC. This is the first molecular analysis in which primary tumors have been examined for the RAD51L1/HMGIC transcripts. The chimeric transcripts were identified from two cases in which exon 7 of the RAD51L1 gene is fused in frame to either exon 2 or exon 3 of the HMGIC gene. These transcripts encode fusion proteins containing RAD51L1 nucleotide binding domains and the HMGIC protein lacking the N-terminal AT hook motifs. The detection of the RAD51L1/HMGIC fusion in primary tumors adds to the accumulating evidence implicating this fusion in a proportion of uterine leiomyoma patients.

A Novel High Mobility Group Protein Gene Is a Candidate for Xp22 Abnormalities in Uterine Leiomyomas and Other Benign Tumors

Because genes of the high mobility group protein family HMGI(Y) are known to take part in the development of a variety of benign solid tumors, the aim of the present study was to search for further members of that family in the human genome. Analysis for HMGI(Y)-related sequences by the polymerase chain reaction (PCR) with the use of cDNA-specific primers offered evidence for HMGIY-like sequences, whereas HMGIC-related sequences were apparently absent. By chromosomal assignment of somatic cell hybrids PCR, HMGIY cDNA-related sequences were detected on seven chromosomes. Positive clones were obtained by screening of a P1-derived artificial chromosome library and mapped by fluorescence in situ hybridization. One of these clones assigned to Xp22.1 was chosen for further analysis because Xp22 is a target region for clonal aberrations in benign solid tumors. Sequence analysis of a DNA fragment of this clone, designated as HMGIYL1, revealed a 94.4% homology to the coding region of HMGIY. Within the HMGIYL1 sequence, no nucleotide sequence divergences leading to a frame shift or a new termination codon compared to HMGIY were found, and a TATA-box-like motif 5′ of it was detected. By reverse transcriptase PCR experiments with the use of HeLa cells and human fetal tissue, HMGIYL1 expression was not detectable. Nevertheless, if not active by itself, it is possible that HMGIYL1 may become activated by chromosomal rearrangements of Xp22 observed in benign solid tumors.

Molecular characterization of a complex chromosomal rearrangement in a pleomorphic salivary gland adenoma involving the 3′-UTR of HMGIC

Recently, the high mobility group protein gene, HMGIC, was identified as a common genetic denominator in benign tumors with chromosome 12q13-15 aberrations, such as lipomas, uterine leiomyomas, pleomorphic adenoma of the salivary glands, hamartomas of breast and lung, angiomyxomas, and endometrial polyps. In most cases, the rearrangements resulted in the separation of the three HMGIC DNA-binding motifs from the acidic carboxy-terminal tail. Here, we report about the molecular characterization of a case of pleomorphic adenoma carrying a t(1;12)(p22;q15). Studies were performed on a cell line derived from the primary tumor, i.e., cell line Ad-312/SV40. Although the chromosome 12 breakpoint was initially mapped more than 1 Mb distal to the HMGIC gene by fluorescence in situ hybridization (FISH) analysis, the present molecular studies reveal a more complex chromosomal rearrangement that directly affects the HMGIC gene. Using 3′-RACE analysis, a HMGIC fusion transcript was detected that contained the complete HMGIC coding region but lacked the putative mRNA destabilizing AUUUA motifs that are normally present in the 3′-UTR of HMGIC. Wild-type HMGIC transcripts were also detected in the tumor cells. The results suggest that alterations in the 3′-noncoding region of HMGIC may have to be considered as pathogenetically relevant.

From chromosome aberrations to the high mobility group protein gene family: Evidence for a common genetic denominator in benign solid tumor development

It is generally accepted that cancer is a genetic disease resulting from the accumulation of somatically acquired genetic changes, sometimes associated with inherited predisposing mutations. In recent years, a variety of cancerrelated genes has been identified; their characterization has already led to significant contributions to diagnosis and to both an understanding of therapy and suggestions for therapies (including gene therapy). It is beyond any doubt that cancer cytogenetics has made historic contributions, since a variety of milestone discoveries in this field started with the molecular analysis of cytogenetically well-defined chromosomal aberrations. In this context, we here discuss benign solid tumors with recurrent aberrations involving chromosome segment 12q13-15. The very first indications implicating this particular chromosome segment in benign solid tumor development came from studies of ordinary lipomas in the laboratory of Dr. Avery Sandberg [1].

Translocations in solid tumours

Specific chromosomal translocations have now been identified in a variety of solid tumour types. Recent developments in this field include the identification of novel genes involved in the t(X;18) synovial sarcoma translocation and the discovery that the high-mobility group protein gene, HMGI-C, is involved in the chromosome 12 rearrangements found in lipomas and uterine leiomyomas. These discoveries may have important implications for the strategies used in tumour diagnosis and treatment.

Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours.

We recently showed that the 1.7 megabase multiple aberration region (MAR) on human chromosome 12q15 harbours recurrent breakpoints frequently found in a variety of benign solid tumours. We now report a candidate gene within MAR suspected to be of pathogenetical relevance. Using positional cloning, we have identified the high mobility group protein gene HMGI-C within a 175 kilobase segment of MAR and characterized its genomic organization. By FISH analysis, we show the majority of the breakpoints of eight different benign solid tumour types fall within this gene. By Southern blot and 3'-RACE analysis, we demonstrate consistent rearrangements in HMGI-C and/or expression of altered HMGI-C transcripts. These results suggest a link between a member of the HMG gene family and benign solid tumour development.