Dinucleotide Deletion Research Articles

Investigating the role of CD24 gene polymorphisms in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease with a complex pathogenesis which involves genetic and environmental factors.1 CD24 is a glycosylphosphatidylinositol-anchored cell surface protein with expression in a wide...

Premature stop codons in a facilitating EF-hand splice variant of Ca V2.1 cause episodic ataxia type 2

Premature stop codons in CACNA1A, which encodes the α 1A subunit of neuronal P/Q-type (Ca V2.1) Ca 2+ channels, cause episodic ataxia type 2 (EA2). CACNA1A undergoes extensive alternative splicing, which contributes to the pharmacological and kinetic heterogeneity of Ca V2.1-mediated Ca 2+ currents. We identified three novel heterozygous stop codon mutations associated with EA2 in an alternately spliced exon (37A), which encodes part of an EF-hand motif required for Ca 2+-dependent facilitation. One family had a C to G transversion (Y1854X). A dinucleotide deletion results in the same premature stop codon in a second family, and a further single nucleotide change leads to a different truncation (R1858X) in a de novo case of EA2. Expression studies of the Y1854X mutation revealed loss of Ca V2.1-mediated current. Because these mutations do not affect the alternate exon 37B, these findings reveal unexpected dependence of cerebellar function on intact exon 37A-containing Ca V2.1 channels.

Identification of a TFIID Binding Sequence That Increases Expression from the Ankyrin-1 Promoter: Application to Globin Gene Therapy

Effective Gene Therapy of the hemoglobin β-chain disorders β-thalassemia or Sickle Cell Disease (SCD), requires that viral vectors deliver β-like globin gene to hematopoietic stem cells (HSC) and express β-globin at levels >20% of that of endogenous α-globin. However, the β-globin gene is poorly expressed without sequences from the Locus Control Region (LCR). The LCR contains sequences that contribute to inefficient production and low titers of the recombinant virus, and pose a significant risk of insertional activation of leukemia genes. We have taken an alternative approach using enhancer independent promoters from genes expressed in erythrocytes to express sufficient β-like globin. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the four different tissue specific promoters used to express the ANK-1 gene. We have shown that a double copy of Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. We have also demonstrated that in mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein were expressed at a uniform level of 7.5% of α-globin per vector copy. To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide in the transcribed region (+4 or +32 relative to the proximal or distal mRNA start sites) of the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We hypothesized that introducing sequences with higher TFIID binding affinity into the region of the ANK-1E promoter would result in increased level of ANK-1E/γ-globin transcription needed for an effective therapy for β-thalassemia and SCD. An ANK-1E promoter library was generated using degenerate sequence in the TFIID binding region while preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained: wild type (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones ((G/T)(G/C)(G/C)GGTGAG) was found at two other locations in the ANK-1E promoter (−5 and −54 relative to the distal mRNA start site) as well as in 25% of 5′UTR across the human genome (p=2.2e−16). 64% of GC rich promoters contain the consensus sequence, which is enriched at +50 and −70 relative to the mRNA start site. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase than the wild type promoter (p=0.001; 0.005 respectively). Electrophoretic mobility shift assays demonstrated that the two more active promoters bind more TFIID than the wild type promoter. We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Mutagenicity of the 1-Nitropyrene-DNA Adduct N-(Deoxyguanosin-8-yl)-1-aminopyrene in Mammalian Cells

The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5'-CCATC GCTACC-3' that has been used for solution NMR investigations. The structural and conformational differences among these lesions are well-established [Patel, D. J., Mao, B., Gu, Z., Hingerty, B. E., Gorin, A., Basu, A. K., and Broyde,S. (1998) NMR solution structures of covalent aromatic amine-DNA adducts and their mutagenic relevance. Chem. Res. Toxicol. 11, 391- 407.]. Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and that the AF adduct was least toxic, with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low-level mutations (<3%) were detected with G-->T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5'-CGC GCG-3' sequence, higher mutational frequency ( approximately 8%) was observed. Again, G-->T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence [Hilario, P., Yan, S., Hingerty, B. E., Broyde, S., and Basu, A. K. (2002) Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene,and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence: A slipped frameshift intermediate model for dinucleotide deletion. J. Biol. Chem. 277, 45068- 45074.]. Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5'-GTGC GTGTTTGT-3' site, the mutations were slightly lower but not very different from the progeny derived from the 5'-CGC GCG-3' sequence. However, the mutational frequency increased by more than 50% when the 5'-C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G-->A, which occurred 2.5 times relative to a 5'-C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5'-C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5' to the adduct. Except for an increase in G-->C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5'-C.

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.

Prevalence of Cholesteryl Ester Storage Disease

Cholesteryl ester storage disease (CESD) is an autosomal recessive chronic liver disease caused by lysosomal acid lipase (LAL) deficiency. The gene is located on chromosome 10q23.2-q23.3, and the enzyme is essential for triglycerides and cholesteryl ester hydrolysis in lysosomes. CESD is characterized by hypercholesterolemia, hypertriglyceridemia, HDL deficiency, and abnormal lipid deposition in many organs. In the liver this results in hepatomegaly caused by hepatic steatosis and fibrosis that can lead to micronodular cirrhosis.1 Disease onset takes place during childhood or adolescence. Males and females are affected in about equal numbers. Patients rarely reach the age of 30. Biochemically, the disorder is recognized by largely reduced lysosomal acid lipase activity.2,3 Complete absence of LAL activity causes Wolman Disease, which is normally fatal within the first 6 months of life.1,4 Several groups have identified mutations in the LAL gene underlying CESD and Wolman disease.5–9 Mutations causing Wolman disease produce an enzyme with no residual activity or no enzyme at all, whereas CESD-causing mutations encode for LAL which retains some enzyme activity.4,10 A G-to-A transition at position −1 of the exon 8 splice donor (E8SJM, E xon 8 S plice J unction M utation) leads to an in-frame deletion of exon 8. The resulting protein is 24 amino acids shorter and has no residual LAL activity, however E8SJM does not cause Wolman Disease because 2% to 4% of normally spliced LAL is present in homozygote carriers.11,12 The vast majority of CESD …

A Dinucleotide Deletion in CD24 Confers Protection against Autoimmune Diseases

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527∼1528 (P1527del) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34–0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22–0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527del allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3′ UTR of CD24 mRNA conveys significant protection against both MS and SLE.

A Destabilizing Di-nucleotide Deletion in 3’-Untranslated Region of Human CD24 mRNA Confers Protection against Multiple Sclerosis (129.1)

Abstract Single-nucleodie polymorphism (SNP) has a profound impact upon one’s susceptibility to autoimmune diseases such as multiple sclerosis (MS). Although it is well established that 3’UTR play a major role in regulating RNA stability, the contribution of SNP in the 3’UTR to the risk of autoimmune diseases has not well characterized. We have proviously reported the contribution of mouse CD24 3’UTR to its mRNA stability, so we investigated the significance of SNPs in the hCD24 3’UTR to MS susceptibility. Our analysis of the 316 MS patients and 342 controls revealed that a di-nucleotide deletion at position 1580 (P1580) of hCD24 mRNA is associated with significantly reduced risk (odds ratio OR = 0.53 with 95% confidence interval = 0.34–0.81) and delayed progression (p = 0.016). More importantly, using 150 families from two independent cohorts, we have found that the P1580del allele is preferentially transmitted to unaffected individuals (p = 0.003). In heterozygous individuals, the mRNA levels for the the di-nucleotide-deletion allele is 2.5-fold less than that of the wild-type allele (p = 0.004). Our transfection studies revealed that the di-nucleotide deletion dramatically reduced the stability of hCD24 mRNA (p &amp;lt; 0.001). Our results demonstrate that a destabilizing di-nucleotide deletion in the 3’UTR of hCD24 mRNA substantially reduces the risk and delays the progression of MS.

Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4 h. The assay works reliably with DNA extracted from hair roots using Chelex-100 ® in a 20 min, 100 °C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.

Skin changes in oculo‐dento‐digital dysplasia are correlated with C‐terminal truncations of connexin 43

Oculo-dento-digital dysplasia (ODDD, OMIM no.164210) is a pleiotropic disorder caused by mutations in the GJA1 gene that codes for the gap junction protein connexin 43. While the gene is highly expressed in skin, ODDD is usually not associated with skin symptoms. We recently described a family with ODDD and palmoplantar keratoderma. Interestingly, mutation carriers had a novel dinucleotide deletion in the GJA1 gene that resulted in truncation of part of the C-terminus. We speculated, that truncation of the C-terminus may be uniquely associated with skin disease in ODDD. Here, we describe a patient with ODDD and palmar hyperkeratosis caused by a novel dinucleotide deletion that truncates most of the connexin 43 C-terminus. Thus, our findings support the notion that such mutations are associated with the occurrence of skin symptoms in ODDD and provide the first evidence for the existence of a genotype-phenotype correlation.

Mutational Analyses of Taiwanese Kindred With X-linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy is a neurodegenerative disorder with highly variable clinical presentation, including the childhood cerebral form, adult form adrenomyeloneuropathy, and Addison disease. The biochemical hallmark of the disorder is the accumulation of saturated very long chain fatty acids in all tissues and body fluids. This accumulation results from mutations in the ABCD1 gene localized to Xq28. Using polymerase chain reaction and direct sequencing of deoxyribonucleic acid, we identified five novel mutations, including a microdeletion (1624 del ATC), a splicing site mutation (intervening sequence 1 [IVS1] -2a>c), and three missense mutations (1172 T>C, 1520 G>A, and 1754 T>C), from Taiwanese kindred with X-linked adrenoleukodystrophy. A polymorphism involving a single nucleotide deletion in the intervening sequence 5 (IVS5 -6 del c) of the ABCD1 gene, previously misattributed as a mutation in the Chinese population, was also identified. The dinucleotide deletion (1415 del AG) mutation common in Japan and Western countries was not found as frequently in the Chinese and Taiwanese populations. Instead, a higher mutation frequency was observed in exon 6 of the ABCD1 gene among Japanese, Chinese, and Taiwanese kindred with X-linked adrenoleukodystrophy, representing a potential mutational hotspot for future mutational screening among these Asian populations.

Functional Characterization of aGJA1Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.

Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 10(4)-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in alpha/beta interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73 %) and amino acid sequence (83 %) identity between ALFV and MVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5'-CU(OH)-3' found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to MVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

Mechanism for N-Acetyl-2-aminofluorene-Induced Frameshift Mutagenesis by Escherichia coli DNA Polymerase I (Klenow Fragment)

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.

Phenotypic Features Associated with Mutations in Steroidogenic Acute Regulatory Protein

Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) are the most common cause of lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by adrenal insufficiency and deficient gonadal steroid synthesis, resulting in female external genitalia in both genetic sexes. We describe three new cases of lipoid CAH caused by novel mutations in the StAR gene. An XY subject of Yemeni descent presented with adrenal insufficiency and severe undervirilization. Magnetic resonance imaging (MRI) of the brain showed enlarged subarachnoid spaces consistent with frontal and temporal atrophy. Two XX siblings of Palestinian descent presented with neonatal adrenal insufficiency. One had a borderline intelligence quotient and features of attention deficit hyperactivity disorder. MRI showed areas of supratentorial white matter lesions. In her sister, MRI revealed a Chiari-I malformation. The XY subject was found to have a missense mutation (R182C). Both XX siblings had a dinucleotide deletion at nucleotides 327-328 that induces a frame shift that truncates the StAR protein after 68 amino acids. These cases broaden the spectrum of known StAR mutations and suggest that disorders of central nervous system development may arise because of StAR deficiency and/or the metabolic consequences of neonatal adrenal deficiency.

Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis

Around 80% of Rett syndrome (RS) cases have a mutation or deletion within the coding sequence of the MeCP2 gene. The other RS patients remain genetically undiagnosed. A significant fraction (10-15%) of disease-causing mutations in humans, affect pre-mRNA splicing. Two potential splice mutations were found in the MeCP2 gene in RS patients, however it was not clear whether these mutations in fact interfere with splicing and consequently cause RS. One such mutation is a deletion of the GT dinucleotide at the 5' donor splice site of exon 1 and the other a deletion of the T nucleotide in the polypyrimidine tract (PPT) of intron 3. Here we experimentally assess the effects exerted by these mutations on the expression of MeCP2 in patients' blood samples and on splicing of the MeCP2 transcript using a hybrid minigene in transient transfection experiments. The results revealed that the Delta T mutation in the PPT is a benign polymorphism and that the GT deletion in intron 1 is a bona fide splicing mutation that causes a complete skipping of exon 1 in the minigene transfection experiment. This explains the observed complete elimination of the MeCP2 message and protein in the lymphoblast clones of the RS patient that carry the mutation on the active X. An analysis of the MeCP2 transcript and protein production in lymphoblast clones, as described here, can be used to confirm clinically diagnosed RS patients with no mutation in the MeCP2 coding sequence. This will enable RS diagnosis without specifically identifying a mutation.

A dinucleotide deletion in the ankyrin promoter alters gene expression, transcription initiation and TFIID complex formation in hereditary spherocytosis

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In some HS patients, mutations in the ankyrin promoter have been hypothesized to lead to decreased ankyrin mRNA synthesis. The ankyrin erythroid promoter is a member of the most common class of mammalian promoters which lack conserved TATA, initiator or other promoter cis elements and have high G+C content, functional Sp1 binding sites and multiple transcription initiation sites. We identified a novel ankyrin gene promoter mutation, a TG deletion adjacent to a transcription initiation site, in a patient with ankyrin-linked HS and analyzed its effects on ankyrin expression. In vitro, the mutant promoter directed decreased levels of gene expression, altered transcription initiation site utilization and exhibited defective binding of TATA-binding protein (TBP) and TFIID complex formation. In a transgenic mouse model, the mutant ankyrin promoter led to abnormalities in gene expression, including decreased expression of a reporter gene and altered transcription initiation site utilization. These data indicate that the mutation alters ankyrin gene transcription and contributes to the HS phenotype by decreasing ankyrin gene synthesis via disruption of TFIID complex interactions with the ankyrin core promoter. These studies support the model that in promoters that lack conserved cis elements, the TFIID complex directs preinitiation complex formation at specific sites in core promoter DNA and provide the first evidence that disruption of TBP binding and TFIID complex formation in this type of promoter leads to alterations in start site utilization, decreased gene expression and a disease phenotype in vivo.

Conservation of promoter, coding and intronic regions of the non‐classical MHC class II DYA gene suggests evolution under functional constraints

The major histocompatibility complex (MHC) in ruminants contains a unique pair of class II genes (DYA and DYB) of unknown function. As functional genes show higher levels of nucleotide conservation than pseudogenes we compared the DYA genes from sheep and cattle, two species which diverged from a common ancestor approximately 20 million years ago. Comparative analysis identified levels of nucleotide conservation in immediate promoter (97%), coding (94%) and intronic regions (91%) comparable with functional MHC genes. The Ovar-DYA transcript revealed an open reading frame encoding a 288 amino acid protein compared with a 253 amino acid protein associated with the BoLA-DYA transcript. A dinucleotide deletion in exon 4 of the Ovar-DYA transcript combined with alternative exon 5 splice sites introduces unusual diversity to the cytoplasmic domain of the Ovar-DYalpha polypeptide. The degree of conservation between these class II MHC genes is consistent with evolution under purifying selection suggesting that these genes retain a unique function in ruminants.

SNP-detecting DNA technologies: Solving problems of applied biochemistry

Heterozygosity of CANP3, ACTN3, and GHR genes in specialized collections was studied using state-of-the-art DNA technologies for DNA analysis. A new dinucleotide deletion (AC) at the beginning of exon 21 was identified in five individuals with heterozygous CANP3 gene. Analysis of polymorphism (SNP1747 C-->T) of ACTN3 gene demonstrated a positive association of allele C with a high muscular performance. Real-time PCR assay of SNP1630 (A-->C) in GHR gene suggested a putative negative association of allele C of this SNP with a high muscular performance.

Mitochondrial Genome Instability and mtDNA Depletion in Human Cancers

An increase in the rate of glycolysis is one of the metabolic alterations in most cancer cells. However, the role of alterations in mitochondrial function and mitochondrial DNA (mtDNA) in carcinogenesis still remains unclear. In this study, we analyzed the nucleotide sequence of the D-loop and the copy number of mtDNA in 54 hepatocellular carcinomas (HCCs), 31 gastric, 31 lung, and 25 colorectal cancers as well as their corresponding non-tumorous tissues. The results revealed that 42.6% (23/54) of the HCCs, 51.6% (16/31) of the gastric cancers, 22.6% (7/31) of the lung cancers, and 40.0% (10/25) of the colorectal cancers harbored mutation(s) in the D-loop of mtDNA. The mtDNA mutations in 43.5% (10/23) of the HCCs, 62.5% (10/16) of the gastric cancers, 57.1% (4/7) of the lung cancers, and 90.0% (9/10) of the colorectal cancers were changes in the mononucleotide or dinucleotide repeats, deletions, or multiple insertions. Moreover, we found that there is a significant decrease in mtDNA copy number in 57.4% (31/54) of the HCCs, 54.8% (17/31) of the gastric cancers, 22.6% (7/31) of the lung cancers, and 28.0% (7/25) of the colorectal cancers compared with the corresponding non-tumorous tissues. It is noteworthy that the incidence of somatic mutations in the D-loop of mtDNA in the cancers of later stages was higher than that of the early-stage cancers. Taken together, our findings suggest that instability in the D-loop region of mtDNA, together with the decrease in mtDNA copy number, is involved in the carcinogenesis of human cancers.