Bp Of Exon Research Articles

Comprehensive Analysis of HLA-G: Implications for Recurrent Spontaneous Abortion

Miscarriage is one of the most common pregnancy complications. Recurrent spontaneous abortion is defined as 2 or more pregnancy losses and may be associated with genetic variation. Human leukocyte antigen-G (HLA-G) is a ligand for natural killer (NK) cell receptors and has the ability to block NK cell activity, which if not blocked can potentially harm a fetus. Consequently a deletion or mutation of the HLA-G gene could lead to miscarriage. Our cases (n = 238) include Caucasian women experiencing 2 or more spontaneous abortions, and controls (n = 233) include women with at least 1 live birth and no history of SA. We sequenced approximately 1400 base pairs (bp) of the HLA-G promoter region, genotyped the 14 bp exon 8 insertion/deletion and single nucleotide polymorphism (SNP) in the coding region of HLA-G. Promoter haplotypes were constructed from sequence information. Twenty-three SNPs were observed in the promoter region with minor allele frequency >0.02. Twelve SNPs differed significantly in frequency between cases and controls. Two haplotypes incorporating these 12 SNPs accounted for 90% of haplotypes and differed significantly in frequency between the 2 populations. Cases were more likely to carry haplotype 2 (P = .0078) and controls to have haplotype 6 (P = .0004). Cases also had a higher frequency of individuals homozygous for the 14 bp insertion. Among the 12 alleles carried on haplotype 2, 5 are predicted to disrupt transcription factor binding sites. The HLA-G promoter is highly associated with the risk of spontaneous abortion, but high linkage disequilibrium in the promoter prevents assignment of the causal variant.

Multiple abnormally spliced ABCA1 mRNAs caused by a novel splice site mutation of ABCA1 gene in a patient with Tangier disease

Background Mutations in ABCA1 gene are the cause of Tangier disease (TD) and familial high density lipoprotein (HDL) deficiency. Splice site mutations of this gene were reported infrequently. Methods ABCA1 gene was sequenced in a TD patient and in subjects with low HDL. The effect of intronic variants on ABCA1 pre-mRNA splicing was studied in COS-1 cells expressing a mutant minigene or in patients' cells. Results A novel mutation in intron 20 (c.2961 –2 A > C) was found in the TD patient. To assess its effect, a mutant ABCA1 minigene, containing intron 18-intron 23 region, was expressed in COS-1 cells. The mutant minigene generated three transcripts: i) in the first (459 bp) 61 nucleotides of intron 20 were retained; ii) in the second (384 bp) exon 20 joined to exon 21 devoid of the first 14 nucleotides; and iii) in the third (255 bp) the entire exon 21 was skipped. The first two transcripts were also observed in patient's peripheral blood mononuclear cells. These mRNAs encode truncated proteins. A variant in intron 8 (c.814 −14 ins A), identified in subjects with low HDL, had no effect on ABCA1 pre-mRNA splicing. Conclusions Functional analysis is required to establish the effect of intronic mutations on ABCA1 pre-mRNA splicing.

Association Between IRF-5 Polymorphisms and Risk of Acute Coronary Syndrome

Previous studies suggested that genetic polymorphisms in interferon regulatory factor 5 (IRF-5) are implicated in the susceptibility to a range of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Recently, IRF-5 has been implicated in inflammatory processes that are associated with excessive remodeling and atherosclerosis. Our purpose was to investigate the association between the IRF-5 polymorphisms and the risk of acute coronary syndrome (ACS) in a Chinese population. The 5 bp indel (insertion/deletion) (CGGGG) polymorphism, located 64 bp upstream of the alternative exon 1a of IRF-5 gene, and the deletion of 30 bp in exon 6 of IRF-5 gene were analyzed among 148 patients with ACS and 246 controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism strategy and direct sequencing. The frequencies of (CGGGG)(3)(CGGGG)(4) genotype and (CGGGG)(4) allele in ACS patients were significantly higher than those in control subjects (p = 0.018, odds ratio [OR] = 1.76, 95% confidence interval [CI]: 1.10-2.81; p = 0.028, OR = 1.62, 95% CI: 1.05-2.50, respectively). However, no significant relationship between the 30 bp exon 6 polymorphism of the IRF-5 gene and the risk of ACS was observed (p = 0.770, OR = 0.96, 95% CI: 0.72-1.28). The 5 bp indel (CGGGG) polymorphism of the IRF-5 gene may be associated with susceptibility to ACS.

An Alternative Splice Variant of KCNMA1 Drives Breast Cancer Metastasis and Invasion.

Abstract Background: We have recently shown that KCNMA1, which encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channel, plays a highly critical role in breast tumor cells metastatic to brain and somewhat less critical role in primary breast cancer cell metastasis. The resulting functional heterogeneity of BKCa channels is attributed generally to alternative splicing of KCNMA1 that generate BKCa channel isoform with altered channel properties. We have identified a KCNMA1 splice variant (KCNMA1vE22) expressed in breast cancer cell line (MDA-MB-361) metastatic to brain with a deletion of 108 bp in exon 22 between the S9 and S10 protein subunit (C-terminus). In this study we investigated the biological function of KCNMA1vE22 in vitro and in vivo.Methods: We validated the results from the breast tumor cell lines data with human normal breast tissue, primary, non-metastatic and metastatic breast tumors by RT-PCR. The KCNMA1vE22 was identified from A-172 tumor cells and cloned into mammalian expression vector (pcDNA6). MCF-7 cells (non-metastatic, null type for KCNMA1vE22) and HEK cells (null type for KCNMA1 and KCNMA1vE22) were transfected with pcDNA6/KCNMA1vE22 and stable clones selected. The expression of KCNMA1 and KCNMA1vE22 was confirmed using RT-PCR, western blot and qPCR. The biological role of the splice variant was studied using parental and transfected MCF-7 cells by cell proliferation, matrigel invasion, transendothelial migration and membrane potential assays in vitro and tumorogenecity using s.c xenograft mouse model.Results: Based on the preliminary screening of breast tumor samples, KCNMA1vE22 was undetectable in primary as well as systemic metastatic breast tumor samples. Stably transfected HEK and MCF-7 cells showed 2.5 to 3-fold increase in KCNMA1 mRNA expression, which corroborated with the increased protein levels and BKCa channel activity. In addition MCF-7 cells expressing KCNMA1vE22 showed a eight-fold increase in invasion while a three-fold increase in TEM was observed compared to parental MCF-7 cells. A marked increase in the proliferation rate of KCNAM1vE22-transfected cells compared to MCF-7 alone was observed, suggesting that KCNAM1vE22 has a profound effect on breast cancer cell proliferation. The in vitro results were validated by subcutaneous mouse model experiment. We found statistically significant increase in mean tumor volume in mice with KCNMA1vE22 transfected compared with parental MCF-7 cells.Conclusions: Our results clearly show that KCNMA1vE22 promotes breast cancer cell invasion, and possibly metastasis to brain. Perhaps the discovery and validation of brain specific metastasis-associated KCNMA1 alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. The variant KCNMA1vE22 that we have discovered potentially may fill the gap to serve as a new generation of biomarker of breast cancer metastasis to brain. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6166.

Non-recurrent SEPT9 duplications cause hereditary neuralgic amyotrophy

BackgroundGenomic copy number variants have been shown to be responsible for multiple genetic diseases. Recently, a duplication in septin 9 (SEPT9) was shown to be causal for hereditary neuralgic amyotrophy...

Extragastrointestinal Stromal Tumors of the Omentum: Review Apropos of a Case with a Novel Gain-of-Function KIT Mutation

We describe a case of extragastrointestinal stromal tumor (EGIST) of the omentum harboring a novel KIT mutation and review the literature on omental EGISTs, emphasizing on molecular genetic data. A large EGIST arising from the lesser omentum was incidentally diagnosed in a 68-year-old man during emergency laparotomy for intra-abdominal hemorrhage following a car accident. The tumor was composed of CD117+/CD34+ spindle-shaped cells with low mitotic activity. Analysis of tumor DNA revealed a heterozygous duplication of 30 bp in exon 11 of KIT. Nine months after R0 resection, positron emission tomography showed abnormal uptake in the upper abdomen. The patient was treated with imatinib mesylate and is alive and well 2 years after the operation. Omental EGISTs remain silent despite a large tumor size. They are diagnosed at a median age of 65 years and show low proliferative activity in the majority (about 80%) of cases. Although the median follow-up period of published cases is only 20 months, mortality appears to be low after R0 resection and is expected to decrease further following the recent introduction of imatinib therapy for high-risk tumors. Accumulating molecular genetic data may lead to improved prognostic classification and patient management.

Lack of Polymorphism in Partial Insulin Like Growth Factor 1 (IGF1) and Insulin Like Growth Factor Binding Protein 3 (IGFBP3) Genes of Mithun

Panigrahi, M., Kumar, S., Deb, S.M., Mitra, A., Sharma, A. and Bujarbaruah, K.M. 2009. Lack of polymorphism in partial insulin like growth factor 1 (IGF1) and insulin like growth factor binding protein 3 (IGFBP3) genes of mithun. J. Appl. Anim. Res., 36: 41–44. A 396 bp fragment encompassing exon 5 of Insulin like growth factor 1 (IGF1) gene and 652 bp fragment encompassing exon 2–3 of Insulin like growth factor binding protein 3 (IGFBP3) gene were digested by HindIII and NlaIII restriction enzymes, respectively, in 90 mithun (Bos frontalis). The PCR-RFLP analysis showed the absence of polymorphism in these fragments in the animals screened. HindIII restriction enzyme produced two fragments of 269 and 127 bp in exon-5 of IGF 1. Similarly NlaIII restriction enzyme produced two fragments of 551 and 101 bp in exon 2–3 ofIGFBP3 gene. Two single nucleotide differences in IGF1 while six such differences in IGFBP3 gene existed in mithun sequences when compared to cattle. The sequence of the amplicons, which were the first reports on these genes in mithun, were submitted to GenBank (Accession numbers EF686016 and EF686017, respectively).

Identification of Distinct Myocardin Splice Variants in the Bladder

Although the importance of myocardin in smooth muscle development is well established, many tissue specific intricacies of smooth muscle differentiation remain to be determined. We characterized myocardin expression in the developing and adult bladder to identify potential tissue specific differences that may have a role in detrusor smooth muscle development. Reverse transcriptase and quantitative polymerase chain reaction were done to determine myocardin expression in the mouse and human bladder vs various other tissues. Sequence analysis was done to confirm the genomic location of the various polymerase chain reaction products. Exonic profiling of the mouse myocardin gene identified a series of unique myocardin splice variants derived from a novel 305 bp exon between exons 2 and 3 of the previously identified myocardin gene. Each variant showed a differential pattern of expression in the mouse and primary protein sequences suggested a unique function for each myocardin variant identified. Identical myocardin splice variants were also observed in the human bladder as well as a unique human specific exon 12 myocardin splice variant that was not observed in the mouse. Identifying a series of unique myocardin splice variants that are differentially expressed in the bladder, and other muscle and nonmuscle tissues provides a potential molecular platform for mediating many unique tissue specific functions associated with the myocardin transcriptional program.

A NewFgf10Mutation in the Mouse Leads to Atrophy of the Harderian Gland and Slit-Eye Phenotype in Heterozygotes: A Novel Model for Dry-Eye Disease?

The purpose of the present study was to characterize a new slit-eye phenotype in the mouse. Genomewide linkage analysis was performed, and a candidate gene was sequenced. Eyes of the mutants were described morphologically, histologically, and by in situ hybridization. To allow morphologic and functional studies of the retina, mutants were outcrossed to C57BL/6. Within an ongoing ethyl-nitrosourea mutagenesis screen with C3HeB/FeJ mice, the authors identified a new mutant (referred to as Aey17) showing a slit-eye phenotype in heterozygotes; homozygous mutants are not viable because of major developmental defects. This mutation was mapped to the distal end of mouse chromosome 13, suggesting Fgf10 (encoding the fibroblast growth factor 10) as a candidate gene. An A-->G transition in the penultimate base of the first intron of Fgf10 leading to aberrant splicing with an additional 49 bp in exon 2 and to a frameshift with a premature stop codon after 54 new amino acids was identified. Histologic analysis of the major ocular tissues (cornea, lens, retina) did not reveal major alterations compared with the wild type, but the size of the Harderian gland was remarkably reduced in heterozygotes. Although Fgf10 was expressed in the developing retina, neither electroretinography nor the virtual drum indicated any abnormalities in heterozygous mutants; overall eye size was identical in wild types and heterozygotes. The mutation in the Fgf10 gene leads to a dominant slit-eye phenotype caused by atrophy of the Harderian gland.

Association of SNPs in exon 2 of the MHC B-F gene with immune traits in two distinct chicken populations: Chinese Beijing-You and White Leghorn

Antibody titers raised for vaccinations against avian influenza (AI) and Newcastle disease (ND) were higher in Chinese Beijing-You (BJY) than in White Leghorn (WL) (P<0.001), but there was no breed difference in titers for sheep red blood cells (SRBC). Genotyping by PCR-SSCP identified seven haplotypes in WL and 17 in BJY. After sequencing PCR products (35 and 85, respectively), 43 (WL) and 47 (BJY) single nucleotide polymorphisms (SNPs) were found in the 264 bp of exon 2. In WL chickens, significant associations were found with antibody responses to AI (two SNPs), ND (six SNPs), and SRBC (one SNP), while in BJY there was association with responses to ND (two SNPs) and SRBC (two SNPs), but none with AI. These results indicate that the genomic region bearing exon 2 of the major histocompatibility complex B-F gene has significant effects on antibody responses to SRBC and vaccination against AI and ND. Different SNPs affected antibody titers for each of the antigens and they differed between these very distinct breeds.

Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA.

Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2) plus 1,476 nt at the 5’ flanking region and 51 nt at the 3’ flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5) to 498 bp (exon 7) and 8 introns from 92 bp (intron 5) to 22 59 bp (intron 1). Furthermore, highly conserved sequences, mainly located in the 5’ flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene) were also identified at 3 different locations of the sequenced region: 5’ untranscribed region, intron 1, and intron 4

Allele-specific fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis (PGD) for 21-hydroxylase deficiency in congenital adrenal hyperplasia (a case report)

OBJECTIVE: Congenital adrenal hyperplasia is a common human genetic disease which in 90% of the cases are caused by a deficiency in the enzyme steroid 21-hydroxylase. Here we describe the implication of a single cell allele-specific PCR for the detection of two mutations in the CYP21 gene. DESIGN: We used the nested PCR method. A direct fragment size analysis of PCR product was performed by fluorescent PCR followed both capillary and conventional gel electrophoresis. MATERIALS AND METHODS: A couple with a previous child died diagnosed for 21-hydroxylase deficiency was referred to our centre. Both parents were carriers of the mutations in CYP21 gene: the mother had the nt656 A/C→G mutation and the father had the deletion of 8 bp in exon 3. Since the father had the deletion of 8 bp in exon 3 and the mother had nt656 A/C→G mutation in intron 2, and the same deletion and mutation always presented in the pseudogene we decided to use in the second round of PCR a primer set consisting of a fluorescently labeled forward primer and reverse primer overlapping the 8 bp deletion region in the father affected allele and in the pseudogene CYP21P. Both primers had a number of nucleotides specified only for CYP21 gene. The using of the strong specific CYP21 gene primers and higher stringency of second round PCR allowed us to avoid amplification of the pseudogene and the father affected allele and amplified only the gene alleles. The total PCR product length was 155 bp. The mother mutation at nt656 was recognized by restriction enzyme digestion using MwoI which cuts the mutated allele into two fragments: 65 bp and 90 bp. The detection of 155 bp fragment in blastomeres would be sufficient to consider them as healthy because of autosomal recessive type of inheritance. RESULTS: The results are in table.Tabled 1nt656 A/C→G (MwoI)D6S1615D6S2894D6S273D6S2920Mother155/90/65126/134338/342126/136198/232Father155/del136/138328/338128/130210/234blast.1155/90/65138/126328/342130/136210/198carrierblast.2155134338126232ADOblast.3155138328130210ADOblast.4155/90/65138/126328/342130/136210/198carrierblast.590/65136/126338/342128/136234/198affectedblast.6155138/124328/338130/126210/232normalblast.7155/90/65138/126328/342130/136210/198carrierblast.8155134338126232ADOblast.9155138/124328/338130/126210/232normal Open table in a new tab CONCLUSIONS: This PGD cycle resulted in a singleton pregnancy followed the healthy girl born.

Variation in the coding and non-coding sequences of the DH-PBAN gene of diapause and non-diapause silkworm races

Diapause hormone (DH) and pheromone biosynthesis-activating neuropeptide (PBAN) code for the diapause gene (DH-PBAN) of Bombyx mori Linnaeus. The DH-PBAN gene sequence from the BLAST database was searched against B. mori genomic gene sequences for similarity. Results showed maximum homology with a genomic contig (accession no. D16230). Primers were designed for the intron, exon and promoter regions of the DH-PBAN gene sequence. The corresponding regions were amplified using genomic DNA as template and products of diapause and non-diapause silkworm races were compared. There was no variation in the 425 bp intron and 254 bp exon regions. However, 1.3% nucleotide sequence variation was observed in selected silkworm races. The promoter region showed 4.2% single nucleotide variation in the diapause and non-diapause silkworm races. The promoter region of the diapause gene showed variations in the sequence of the POU (Pit 1, Oct 1 and Unc-86)-binding site of non-diapause and diapause races of B. mori. In phylogenetic analysis, B. mori formed a cluster with other silkworms of the Bombycidae family, whereas the Noctuidae family formed a separate cluster, indicating that the diapause gene is conserved across the insect taxa.

ATR alterations in Hodgkin's lymphoma

Hodgkin's lymphoma (HL) is characterized by the presence of neoplastic Hodgkin and Reed-Sternberg cells (HRSC) in a background of inflammatory cells. Free radicals and oxidative stress generated in the inflammatory lesions could cause DNA damage, thus providing a basis for lymphomagenesis. Ataxia-telangiectasia mutated (ATM) and Rad3-related (ATR) genes are responsive genes for DNA damage, therefore the potential involvement of the ATR gene in HL pathogenesis was examined in 8 HL cell lines and 7 clinical cases. ATR alterations were detected in 6 out of 8 HL lines. Most aberrant transcripts observed were heterozygous deletions, which may have resulted from aberrant splicing. ATR aberrant transcripts were also detected in 3 out of 7 clinical cases. Three alterations, del exon 4, deletion exon 29-34 and insertion of 137 bp in exon 46/47 were commonly observed in both cell lines and clinical samples. HL cells with ATR alterations except del exon 4 showed a delay/abrogation in repair for DNA double-strand breaks (DSBs) and single-strand break (SSB) as well as exhibiting a defect in p53 accumulation. These findings suggested the role of ATR gene alterations in HL lymphomagenesis.

SRp20 and hnRNP A1 binding to IR exon 11 modulate its alternative splicing

Alternative splicing of the insulin receptor (IR) transcripts generates two isoforms: isoform A (IR‐A), which lacks 36 bp exon 11, and isoform B (IR‐B), which includes exon 11. The IR‐B and IR‐A isoforms show a cell type specific distribution with their relative proportion varying during development, aging and different disease states. Normally, IR‐B predominates in insulin‐sensitive tissues such as liver, muscle, adipocytes and kidney whereas, various pathological conditions mostly express IR‐A isoform. Also, IR‐B has greater insulin responsiveness than does IR‐A. Recent studies demonstrate that IR gene also exhibits metabolic and hormonal regulation of splicing, although the regulatory factors are mostly unknown. Our lab has previously demonstrated that both exon 11 and intron 10 contain regulatory sequences that affect IR alternative splicing both positively and negatively. Here, we focus on the role of specific cis acting regulatory elements within exon 11 of the IR gene. Fine mapping of cis acting elements within exon has been carried out by six linker scanning mutations (LS1‐LS6) throughout the exon 11. Results indicate the presence of an exonic splicing enhancer (ESE) in the first 12 nucleotides and an exonic splicing silencer (ESS) within the central 12 nucleotides of exon 11. Most naturally occurring ESEs have been shown to bind specific SR proteins, whereas the best characterized ESSs are bound by particular hnRNP proteins. Sequence analysis of ESE in IR exon 11 revealed that it contains a (CUCUUC) consensus binding sequence for SR protein, SRp20. The ESS contains a sequence (CAGGCA) that partially matches the previously identified high‐affinity binding sequence for hnRNP A1 (UAGGGA). Co‐transfection in HepG2 cells of IR minigene B and several SR and hnRNP protein expression vectors reveals that hnRNP A1 is a negative regulator, whereas SRp20 is a positive regulator for IR exon 11 inclusion. Co‐transfection of the deletion and linker scanning mutants of exon 11 with SRp20 or hnRNP A1 confirmed the binding location of these proteins in IR exon 11. To establish in‐vitro binding of SRp20 and hnRNP A1 in ESE and ESS respectively, an RNA affinity assay was performed using RNA templates corresponding to wild type and linker scanning mutants in exon 11, coupled to adipic acid beads and incubated with HeLa cell nuclear extract. By western blot analysis of the eluted proteins, we have established that ESE binds SRp20 whereas the ESS binds hnRNP A1. Co‐transfection of minigene B with siRNA against hnRNP A1 increased whereas siRNA against SRp20 decreased the inclusion of exon 11. Purification of these regulatory proteins for in‐vitro splicing assay as well as UV cross linked competition assay using radio‐labeled probes is currently underway. These results demonstrate for the first time that SRp20 and hnRNP A1 play an antagonistic regulatory role in IR alternative splicing via binding to juxtaposed exonic elements.

Mutation Analysis of the VMD2 Gene in Thai Families with Best Macular Dystrophy

Background: To identify genetic mutations of the VMD2 gene in two Thai families with Best macular dystrophy. Materials and Methods: Ophthalmic examination including best-corrected visual acuity (BCVA), dilated fundus examination and fundus photography, and electro-oculography (EOG) was performed in two probands and their family members. Mutation screening of the VMD2 gene was performed by single-strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing of the abnormal exons. Results: The 58-year-old male proband demonstrated typical egg yolk-like macular lesion in both eyes. Mutational screening of VMD2 identified a band shift in exon 7, which was confirmed by direct DNA sequencing to be a G to A transition at position 724 bp. This novel missense mutation resulted in the change of an amino acid valine to methionine and was responsible for the abnormal Arden ratio in the proband's daughter. The second male proband age 25 had a characteristic egg yolk-like macular lesion in the left eye and a scrambled egg appearance in the right. Mutation analysis identified a C to T transition at position 652 bp in exon 6. This reported missense mutation led to an amino acid substitution of cysteine for arginine. The mutation was documented in the maternal grandmother, the mother, as well as the elder sister of the proband. Conclusions: The Val-242-Met mutation is associated with a late-onset visual disturbance and the Arg-218-Cys mutation was associated with marked intra-familial clinical variability of expression. Presymptomatic testing will be available to the family members at risk with high accuracy.

CD55−CD59− Granulocytes in Patients with Aplastic Anemia Are Clonal Populations Derived from Single PIG-A Mutant Stem Cells without Any Proliferative Advantage.

Small populations of CD55−CD59− blood cells are detectable in approximately 50% of all acquired aplastic anemia (AA) patients and the presence of such PNH-type cells are also associated with a good response to immunosuppressive therapy (Sugimori C, et al. Blood 2006). In most patients showing 0.1% to 1.0% PNH-type cells at the diagnosis of AA, the PNH-type cell proportion remains unchanged over 3 years even after successfully responding to immunosuppressive therapy (Mochizuki K, et al. ASH 2006). Although these findings suggest that small populations of PNH-type cells are derived from a limited number of PIG-A mutants without any proliferative advantage, this hypothesis has not yet been verified at the molecular level. To appropriately address this issue, we studied 3 patients with AA who showed 0.14 to 1.6% PNH-type granulocytes. The CD55−CD59− granulocytes were sorted from these patients 2 different times at a minimum of 6 month intervals and then they were subjected to a PIG-A gene analysis. Five exons were amplified using 6 different primer sets and each amplified product was then subcloned into E. coli. At least 5 transformed clones for each amplified product were randomly plucked and subjected to sequencing. Single mutations were thereafter detected in all 3 patients as shown in Table 1. The same single mutations were then detected in the CD55−CD59− granulocytes from the patient obtained 6 months after the first examination. Two patients were in a state of hematologic remission at from 1 to 7 years after the first examination of their peripheral blood and the proportion of PNH-type cells remained stable during this period in all patients. Response to immunosuppressive therapy was not evaluable in one patient because he rejected ATG therapy. These findings indicate that PNH-type granulocytes from patients with AA are therefore clonal populations derived from single hematopoietic stem cells (HSCs) with a PIG-A mutation. If an AA patient has many HSCs with PIG-A mutations before the development of AA, then the immune system attack against HSCs should allow for the survival of the PIG-A mutants leading to the generation of a polyclonal PNH-type cell population. The presence of clonal PNH-type cells at the time of AA diagnosis suggests that the number of HSCs with a PIG-A mutation in healthy individuals may therefore be much lower than we expected and the paucity of PIG-A mutant HSCs may therefore account for the absence of increased number of PNH-type cells in approximately 20% of all AA patients who apparently respond to immunosuppressive therapy.Table 1PatientAgeGenderProportion of PNH-type granulocytesPIG-A mutationResponse to IST164M0.147%593 bp (exon 2)T insertionPR282M1.629%3′splice site (intron 1)G to A changePR376M0.161%276 bp (exon 2)G deletionnot evaluable

Novel Spliced MLL Fusions Have Been Identified Involving the MLL Partner Genes ELL, EPS15, MLLT3, and SEPT5.

Chromosomal rearrangements of the human MLL gene are a genetic hallmark for aggressive acute leukemias. More than 60 partner genes have been characterized at the molecular level until now. The observed recombination mechanisms between MLL and its many recombination partner genes include reciprocal chromosomal translocations, insertions, and intrachromosomal 11q deletions and inversion. “Spliced MLL fusions”, have recently been described as a novel mechanism to generate functional chimeric fusion transcripts for 50% of t(11;19)(q23;p13.3) leukemia patients where the MLL gene is fused to the MLLT1 (ENL) gene and for two single leukemia patients. One patient had a t(11;15) involving the ZFYVE19 gene and the other patient had a 11q interstitial deletion involving the DCPS gene. Within these cases the disrupted MLL gene is fused only on RNA level to a functional transcription unit located in the vicinity of the breakpoint. Here we describe eight novel “spliced MLL fusions” involving the MLL partner genes ELL (1x), EPS15 (3x), MLLT3 (1x), and SEPT5 (1x). For ELL, MLLT3 and SEPT5 the identified spliced fusion seems to be a single or rare event. However 1/3 of the t(1;11)(p32;q23) leukemia cases exhibit “spliced MLL fusions”. I.e. approximately 1/3 of all breakpoints are located upstream of the EPS15 gene. Further studies will help to investigate the remaining question whether these two groups of t(1;11)(p32;q23) leukemia patients have any significant difference in outcome. Interestingly, for the t(11;22)(q23;q11.2) leukemia the major identified fusion transcript has an additional 57 bp exon derived from the 2 kb area up-stream of the SEPT5 gene. Furthermore this patient has a T-ALL and thus differs from all other cases where the patients show myeloid markers when the MLL gene is fused to one of the five members of the septine family. This is leading to the question whether or not this 57 bp big exon can be made responsible for the lineage shift. With these results the number of genes involved in „spliced MLL fusions” has increased from three to seven. Supported in part by grants Ma 1876/7-1, /8-1 and /9-1 from the DFG, grant N1KR-S12T13 from the BMBF and grant 102362 from the Deutsche Krebshilfe.

Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

BackgroundGhrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0). The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus.ResultsWe have demonstrated the presence of an additional novel exon (exon -1) and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin) and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand) transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons.ConclusionThe sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin. This study reveals several novel aspects of the ghrelin gene and suggests that the ghrelin locus is far more complex than previously recognised.