TATA Box Binding Protein Gene Research Articles

Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies.

Reference genes are used as internal reaction controls for gene expression analysis, and for this reason, they are considered reliable and must meet several important criteria. In view of the absence of studies regarding the best reference gene for the analysis of acute leukemia patients, a panel of genes commonly used as endogenous controls was selected from the literature for stability analysis: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Abelson murine leukemia viral oncogene human homolog 1 (ABL), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Ribosomal protein lateral stalk subunit P0 (RPLP0), β-actin (ACTB) and TATA box binding protein (TBP). The stability of candidate reference genes was analyzed according to three statistical methods of assessment, namely, NormFinder, GeNorm and R software (version 4.0.3). From this study's analysis, it was possible to identify that the endogenous set composed of ACTB, ABL, TBP and RPLP0 demonstrated good performances and stable expressions between the analyzed groups. In addition to that, the GAPDH and HPRT genes could not be classified as good reference genes, considering that they presented a high standard deviation and great variability between groups, indicating low stability. Given these findings, this study suggests the main endogenous gene set for use as a control/reference for the gene expression in peripheral blood and bone marrow samples from patients with acute leukemias is composed of the ACTB, ABL, TBP and RPLP0 genes. Researchers may choose two to three of these housekeeping genes to perform data normalization.

Mutation analysis of the TATA box-binding protein (TBP) gene in Russian patients with spinocerebellar ataxia and Huntington disease-like phenotype

ObjectiveWe aimed to analyze the occurrence and clinical and genetic characteristics of spinocerebellar ataxia type 17 (SCA17) among Russian patients with progressive cerebellar ataxia or Huntington disease-like phenotype. MethodsGenetic analysis of CAG/CAA repeats in TBP gene was carried out in 217 patients, including 153 patients with progressive unspecified ataxia and 64 patients with Huntington disease-like phenotype. SCA types 1, 2, 3, 6 and 8, Friedreich's ataxia, CANVAS and Huntington disease were preliminarily excluded. ResultsSix unrelated patients with SCA17 (2.8 %) were identified (43–57 CAG/CAA repeats in TBP gene). Two patients had a positive family history. Age at the disease onset ranged from 15 to 47 years. The core clinical syndrome included progressive cerebellar ataxia, dysarthria, movement disorders, cognitive impairment, and psychiatric symptoms. One patient had epilepsy with rare generalized tonic-clonic seizures. Another patient with diffuse muscle atrophy and small expansion size (43 CAG/CAA repeats) had myopathic changes in skeletal muscles on EMG study. We also described a patient with a large expansion size of 57 CAA/CAG repeats with early onset and rapid disease progression. ConclusionSCA17 is a relatively rare cause of progressive disorders with ataxia and chorea, but it should be considered in the spectrum of differential diagnosis in such patients. Most of our SCA17 cases were sporadic which should be kept in mind when planning genetic testing in patients with spinocerebellar ataxia and chorea.

Small-expanded allele spinocerebellar ataxia 17: imaging and phenotypic variability.

Spinocerebellar ataxia 17 (SCA17) is a rare genetic cause of adult-onset ataxia caused by an abnormal expansion of the CAG/CAA sequence in the TATA-box Binding Protein (TBP) gene. A number of repeats higher than 49 are full penetrance-expanded. The range between 41 and 49 repeats is characterized by decreased penetrance, and it is usually referred to as "small." Here, we describe two patients with the SCA17 phenotype and with 43 and 44 CAG repeats in the TBP gene, and review all the previously reported cases of SCA17 with a small range of expansions. We focus on both clinical features and imaging findings, which, in the case of small-expanded alleles, can resemble those of atypical parkinsonisms. Thus, we suggest to consider the small-expanded allele SCA17 as a possible diagnosis in patients with adult-onset ataxia, even when both clinical and imaging characteristics are suggestive for other non-genetic neurodegenerative diseases.

Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes.

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

Investigating TBP CAG/CAA trinucleotide repeat expansions in a Taiwanese cohort with ALS

Intermediate-length CAG repeats in ATXN2 have been well recognized as a genetic risk factor for amyotrophic lateral sclerosis (ALS). However, the role of similar trinucleotide repeat expansions in the TATA-box binding protein gene (TBP), another disease-associated gene for inherited ataxia, in ALS remains elusive. To assess the association between TBP trinucleotide repeat expansions and ALS, we investigated the repeat lengths in 325 unrelated ALS patients and 1500 controls in the Taiwanese population. The most common size of repeats in the patients and controls were both 36. The repeat lengths ranged from 29 to 46 repeats in the ALS patients and 27 to 43 repeats in the controls. Two ALS patients carried a TBP allele with a repeat number equal or greater than 44 (44 and 46). The patient with the 46 trinucleotide repeats also had a C9ORF72 GGGGCC hexanucleotide repeat expansion. The odds ratio of an individual carrying the CAG/CAA repeats ≥ 44 to have ALS is 23.2 (95% confidence interval: 1.11–484.24; p = 0.04). Our findings suggest that intermediate-length CAG/CAA repeat expansions in TBP may associate with ALS risk.

Complexity of the Genetics and Clinical Presentation of Spinocerebellar Ataxia 17.

Spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the TATA-box binding protein gene (TBP). The disease has a varied age at onset and clinical presentation. It is distinct from other SCAs for its association with dementia, psychiatric symptoms, and some patients presenting with chorea. For this reason, it is also called Huntington’s disease-like 4 (HDL-4). Here we examine the distribution of SCA17 allele repeat sizes in a United Kingdom-based cohort with ataxia and find that fully penetrant pathogenic alleles are very rare (5 in 1,316 chromosomes; 0.38%). Phenotype-genotype correlation was performed on 30 individuals and the repeat structure of their TBP genes was examined. We found a negative linear correlation between total CAG repeat length and age at disease onset and, unlike SCA1, there was no correlation between the longest contiguous CAG tract and age at disease onset. We were unable to identify any particular phenotypic trait that segregated with particular CAG/CAA repeat tract structures or repeat lengths. One individual within the cohort was homozygous for variable penetrance range SCA17 alleles. This patient had a similar age at onset to heterozygotes with the same repeat sizes, but also presented with a rapidly progressive dementia. A pair of monozygotic twins within the cohort presented 3 years apart with the sibling with the earlier onset having a more severe phenotype with dementia and chorea in addition to the ataxia observed in their twin. This appears to be a case of variable expressivity, possibly influenced by other environmental or epigenetic factors. Finally, there was an asymptomatic father with a severely affected child with an age at onset in their twenties. Despite this, they share the same expanded allele repeat sizes and sequences, which would suggest that there is marked difference in the penetrance of this 51-repeat allele. We therefore propose that the variable penetrance range extend from 48 repeats to incorporate this allele. This study shows that there is variability in the presentation and penetrance of the SCA17 phenotype and highlights the complexity of this disorder.

Validation of expression stability of reference genes in response to herbicide stress in wild oat (Avena ludoviciana)

Weeds are serious problem in crop production and wild oat is a grass weed of economic and agronomic significance. We need to extend our basic knowledge of weeds especially in molecular genetics and gene expression. For study of gene expression by semi-quantitative and quantitative PCR, it is recommended that normalization of reference genes be carried out in order to select the most stable reference gene for a precise gene expression study. The purpose of this research was evaluation of four reference genes in response to treated and untreated (control) by herbicide in two tissues (stem and leaf) of non-target site resistance wild oat (A. ludoviciana). Four candidate reference genes including Actin, Ef1α (elongation factor 1 alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and TBP (TATA-box-binding protein) were used to determine stable reference gene exposed to the herbicide using the statistical methods of NormFinder, BestKeeper and delta-Ct. NormFinder indicated that TBP and Actin genes are the best combination of two genes for normalizing calculations (with a combined gene stability value of 0.012) for qPCR analysis under herbicide stress in different tissues of non-target site resistance wild oat. Based on the statistical results, the Ef1α gene was identified as the unstable reference gene. Totally, according to results of this study, TBP gene is the most stable reference gene and therefore, this gene can be used as a reference gene for future studies of quantitative PCR analysis of herbicide stress-responsive gene expression in wild oat and potentially in other grass weed species.

Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition.

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, we identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, we found that TRF2 and TAF9 affects the transcription of a common set of genes, including peroxisomal fatty acid β-oxidation-related genes that affect phospholipid fatty acid composition. We also found that knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis.

Downregulation of proteins involved in the endoplasmic reticulum stress response and Nrf2-ARE signaling in lymphoblastoid cells of spinocerebellar ataxia type 17

Spinocerebellar ataxia type 17 (SCA17) is caused by CAG repeat expansion in the TATA-box binding protein gene. Studies of several polyglutamine (polyQ) expansion diseases have suggested that the expanded polyQ proteins misfold and induce oxidative stress to contribute to cell death. Substantial deficits in peripheral tissues including lymphocytes have been shown and these peripheral abnormalities could also be found in neurons possessing polyQ disease proteins. In this study, we used a lymphoblastoid cell model to investigate the functional implication of SCA17 expanded alleles and assess the potential therapeutic strategies that may ameliorate the effects of expanded polyQ. Proteomics studies of patient/control pairs including two-dimensional (2-D) gel electrophoresis, mass spectrometry and immunoblotting were conducted. A total of 8 proteins with reduced expression changes greater than 1.3-fold were identified, including previously reported HSPA5 and HSPA8. Among 6 proteins further semi-quantified by immunoblotting and real-time PCR, the reduced expression of HYOU1, PDIA3, P4HB, NQO1 and HMOX1 was confirmed. Treatment with resveratrol and genipin up-regulated NQO1 and HMOX1 expression and reduced oxidative stress in patients' lymphoblastoid cells. The results illustrate downregulation of proteins involved in the endoplasmic reticulum stress response (HYOU1, HSPA5, PDIA3, and P4HB) and Nrf2-ARE signaling (NQO1 and HMOX1) in SCA17 lymphoblastoid cells. Compounds increasing anti-oxidative activity such as resveratrol and genipin may serve as a potential therapeutic strategy for SCA17.

Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs.Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan® assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms.Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes.For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended.

A novel transgenic rat model for spinocerebellar ataxia type 17 recapitulates neuropathological changes and supplies in vivo imaging biomarkers.

Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.

From Normal Gait to Loss of Ambulation in 6 Months: a Novel Presentation of SCA17

Spinocerebellar ataxias are a group of rare and heterogeneous autosomal dominant disorders characterized by progressive ataxia and other features. Spinocerebellar ataxia 17 (SCA17) is one of the 32 subtypes described to date and is secondary to CAG/CAA repeat expansion in the gene coding for the TATA-box binding protein (TBP). SCA17 is clinically heterogeneous and typically presents with slowly evolving ataxia, dysarthria, dementia, depression, and other movement disorders such as chorea. More than 41 CAG/CAA repeats are considered diagnostic of SCA17, with more than 49 being associated with full penetrance. We report one patient presenting with isolated rapidly evolving ataxia who was found to have 44 CAG/CAA repeats in the TBP gene. This suggests that, while SCA17 typically slowly progresses over years, its repertoire of presentations should be expanded to include rapidly progressive isolated ataxia resembling paraneoplastic disorders or prion disease.

A case of nocturnal frontal lobe epilepsy in a patient with spinocerebellar ataxia type 17

Nocturnal frontal lobe epilepsy (NFLE) comprises a complex spectrum of distinct paroxysmal sleep-related attacks of variable intensity, including paroxysmal arousals, nocturnal paroxysmal dystonias and epileptic nocturnal wanderings. Scalp EEG and brain MRI are unable to reveal abnormalities in a considerable amount of patients. In those cases the epileptic nature of NFLE is supported by the stereotypical clinical presentation of the episodes and the often dramatic response to antiepileptic drugs (AEDs) like Carbamazepine, Clonazepam or Topiramate. Spinocerebellar ataxia type 17 (SCA 17) is a neurodegenerative disease caused by the expansion above 44 units of a CAG/CAA repeat in the coding region of the TATA box binding protein (TBP) gene. It is one of the rarer forms amongst autosomal dominant cerebellar ataxias, with extreme variable age at onset and clinical phenotype. Epilepsy is quite commonly observed among SCA 17 patients, however it is exclusively described in terms of generalized tonic–clonic seizures. We report a case of NFLE occurring in a SCA 17 patient and brilliantly responsive to Topiramate after failure of the first line therapy with Carbamazepine.

C10 Generation and characterisation of a transgenic rat model of huntington's disease-like 4 (HDL4), also called spinocerebellar ataxia type 17 (SCA17)

Huntington9s disease-like 4 (HDL4), also called spinocerebellar ataxia type 17, is an autosomal-dominant, late-onset neurodegenerative disorder caused by CAG repeat expansions in the TATA-box-binding protein (TBP), an ubiquitously expressed transcription factor. The clinical features of HDL4 closely resembled those of Huntington9s disease, including uncontrolled movements, emotional problems, and loss of thinking ability. To further investigate this devastating disease and additionally find a good model for treatment studies, we generated the first transgenic rat model of HDL4, which carries a full human cDNA fragment of the TBP gene with 64 CAG repeats under the control of the murine prion protein promoter (PrP-TBP64Q). In total we obtained ten positive founder animals, whereof only five transmitted the transgene. On the basis of the expression level of mutant TBP as well as the distribution of mutant TBP in the different brain regions we chose one of these five lines (line 8.4) for further characterisation. This line shows a strong expression of the mutant TBP protein in the cerebellum and a moderate expression in the olfactory bulb, brainstem and cortex. HDL4 rats showed an ataxia-like phenotype and impaired motor coordination and balance capabilities in the beam walking test. Additionally, HDL4 rats had a significantly lower body weight than their wildtype littermates and showed decreased activity. At the age of 10 months neuropathological changes, such as misshaped Purkinje cells, degenerated dendrites as well as missing basket and stellate neurons were observed in the cerebellum of heterozygous transgenic HDL4 rats compared to wildtype littermates. By electron microscopy we demonstrated dark cell degeneration as well as moderate fibre degeneration in the striatum. Moreover, we observed decreased levels of dopamine receptor (D₂R) by performing PET imaging. Altogether the present data shows that this animal model exhibits symptoms which mimic well the human HDL4 phenotype indicating that this rat model will be of great value for further studies of pathomechanisms in Huntington9s disease-like 4 and for therapeutic trials.

Severe and rapidly progressing cognitive phenotype in a SCA17-family with only marginally expanded CAG/CAA repeats in the TATA-box binding protein gene: A case report

BackgroundThe autosomal dominant spinocerebellar ataxias (SCAs) confine a group of rare and heterogeneous disorders, which present with progressive ataxia and numerous other features e.g. peripheral neuropathy, macular degeneration and cognitive impairment, and a subset of these disorders is caused by CAG-repeat expansions in their respective genes. The diagnosing of the SCAs is often difficult due to the phenotypic overlap among several of the subtypes and with other neurodegenerative disorders e.g. Huntington’s disease.Case presentationWe report a family in which the proband had rapidly progressing cognitive decline and only subtle cerebellar symptoms from age 42. Sequencing of the TATA-box binding protein gene revealed a modest elongation of the CAG/CAA-repeat of only two repeats above the non-pathogenic threshold of 41, confirming a diagnosis of SCA17. Normally, repeats within this range show reduced penetrance and result in a milder disease course with slower progression and later age of onset. Thus, this case presented with an unusual phenotype.ConclusionsThe current case highlights the diagnostic challenge of neurodegenerative disorders and the need for a thorough clinical and paraclinical examination of patients presenting with rapid cognitive decline to make a precise diagnosis on which further genetic counseling and initiation of treatment modalities can be based.

Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress

Heat stress decreases immune function and increases disease susceptibility in stressed animals, which are important factors for industry and public health. We investigated the molecular mechanisms underlying heat stress by profiling the expression of target genes involved in the cellular response in the blood of Bama miniature pigs ( Sus scrofa domestica) over 21 days with the use of quantitative RT-PCR (qRT-PCR). Reliable standards were established for the normalization of qRT-PCR. Six potential reference genes were ranked by their stability using the geNorm and NormFinder programs. Ribosomal protein L4 ( RPL4) and TATA-box-binding protein ( TBP) ranked as the two most stably expressed genes, except on day 21 when beta-2-microglobulin ( B2M) was the most stable. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and ribosomal RNA 18S ( 18SRNA) were discarded as reference genes due to their unstable expression patterns. When B2M and TBP genes were selected as standards in combination, rather than GAPDH, a significant upregulation in Toll-like receptor 2 ( TLR2) expression was observed after 21 consecutive days of heat stress. These findings suggest that selection of an ideal reference gene is a key step in comparison of transcription profiles in Bama miniature pigs.

Neuronal expression of TATA box-binding protein containing expanded polyglutamine in knock-in mice reduces chaperone protein response by impairing the function of nuclear factor-Y transcription factor

The polyglutamine diseases consist of nine neurodegenerative disorders including spinocerebellar ataxia type 17 that is caused by a polyglutamine tract expansion in the TATA box-binding protein. In all polyglutamine diseases, polyglutamine-expanded proteins are ubiquitously expressed throughout the body but cause selective neurodegeneration. Understanding the specific effects of polyglutamine-expanded proteins, when expressed at the endogenous levels, in neurons is important for unravelling the pathogenesis of polyglutamine diseases. However, addressing this important issue using mouse models that either overly or ubiquitously express mutant polyglutamine proteins in the brain and body has proved difficult. To investigate the pathogenesis of spinocerebellar ataxia 17, we generated a conditional knock-in mouse model that expresses one copy of the mutant TATA box-binding protein gene, which encodes a 105-glutamine repeat, selectively in neuronal cells at the endogenous level. Neuronal expression of mutant TATA box-binding protein causes age-dependent neurological symptoms in mice and the degeneration of cerebellar Purkinje cells. Mutant TATA box-binding protein binds more tightly to the transcription factor nuclear factor-Y, inhibits its association with the chaperone protein promoter, as well as the promoter activity and reduces the expression of the chaperones Hsp70, Hsp25 and HspA5, and their response to stress. These findings demonstrate how mutant TATA box-binding protein at the endogenous level affects neuronal function, with important implications for the pathogenesis and treatment of polyglutamine diseases.

Ophthalmologic abnormalities in a de novo terminal 6q deletion

Purpose: To correlate the clinical phenotype with the genotype of a boy with a terminal deletion of chromosome 6q and to compare these observations to previous reports of 6q deletions and review of the literature.Methods: Careful clinical evaluation, conventional cytogenetic analysis on GTG-banded chromosomes and 244K array CGH analysis.Results: This 14 year old Saudi boy had modest mental retardation, seizures, microcephaly, cortical dysplasia, a non-comitant esotropia, impersistent eccentric gaze, congenital nystagmus, thick corneas, and substantial myopia. He had a de novo 10.79 Mb deletion on chromosome 6 from 6q25.3 to 6qter. The deleted region extended from nucleotide 159929512 to 170723629 and encompassed 87 genes. Eleven genes remained within the proband’s deleted region after excluding genes located in deleted areas reported in phenotypically normal individuals. Among those 11 genes, only the TBP (TATA box binding protein) gene has been associated with any symptom or sign observed in our patient.Conclusions: This boy had clinical features similar to patients reported with the 6q terminal deletion syndrome. In addition, he had an unusual ocular motility pattern and thick corneas, features that may be more common than previously recognized. Deleted genes in this area of chromosome 6 may contribute to ophthalmic abnormalities in addition to mental retardation.

SCA17 repeat expansion: Mildly expanded CAG/CAA repeat alleles in neurological disorders and the functional implications

Spinocerebellar ataxia type 17 (SCA17) involves the expression of a CAG/CAA expansion mutation in the gene encoding TATA-box binding protein (TBP), a general transcription initiation factor. The spectrum of SCA17 clinical presentation is broad. We screened for triplet expansion in the TBP gene in Taiwanese Parkinson's disease (PD), Alzheimer's disease (AD) and atypical parkinsonism and investigated the functional implication of expanded alleles using lymphoblastoid cells as a model. A total of 6 mildly expanded alleles (44-46) were identified in patients group. The frequency of the individuals carrying expanded alleles in PD (3/602 [0.5%]), AD (2/245 [0.8%]) and atypical parkinsonism (1/44 [2.3%]) is not significant as compared to that in the control subjects (0/644 [0.0%]). In lymphoblastoid cells, HSPA5, HSPA8 and HSPB1 expression levels in cells with expanded TBP were significantly lower than that of the control cells. Although not significantly, the levels of PARK7 protein isoforms 6.1 and 6.4 are notably increased in SCA17 lymphoblastoid cells. Treatment of TBH (tert-butyl hydroperoxide) significantly increases cell death in the cells with mildly expanded TBP. Our findings expand the spectrum of SCA17 phenotype and may contribute to our understanding of the disease.

Validation of Putative Reference Genes for Normalization of Q-RT-PCR Data From Paraffin-embedded Lymphoid Tissue

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.