Conformation Sensitive Gel Electrophoresis Research Articles

A novel BRCA1 mutation in an Indian family with hereditary breast/ovarian cancer

Germ-line mutations in BRCA1 gene contribute to a majority of familial breast and ovarian cancers. A group of 23 Tamil Nadu (south India) patients with positive family history for breast and ovarian cancer were screened for BRCA1 mutations by conformation sensitive gel electrophoresis (CSGE) followed by sequencing. In the present study, we report a novel 1307delT mutation in exon 11 of BRCA1 gene in a 43-year-old woman of Indian origin with breast cancer. This mutation gives rise to a premature stop codon at amino acid residue 409 and also creates a novel DdeI restriction site. The same mutation was also detected in the patient's maternal uncle and his son through extended family analysis. The 1307delT is a novel mutation that has not been documented in any population or published report to the best of our knowledge. Identification of this novel mutation stresses the need for developing a database of BRCA1 mutations, which will aid in breast cancer screening in this population.

Acquired Mutations in the BCR-ABL Kinase Domain in Imatinib Resistant Patients. Multicentric Experience in Argentina and Uruguay.

Imatinib induces complete cytogenetic response (CCyR) in 82% of patients with CML in chronic phase. There is a subset of patients who either failed to achieve or lose hematological/CCyR. Kinase domain mutations have emerged as a potential cause for treatment failure.Aims: In the current study we have investigated ABL kinase domain mutations, amplifications and quantification of BCR-ABL transcripts by Q-PCR in Imatinib resistant patients.Patients and Methods: A total of 84 patients (pts) with CML treated with Imatinib were studied and 82 were evaluable. Sixty-one (74%) were in chronic phase (CP), eleven (14%) were in accelerated phase (AP), and ten (12%) were in blast crisis (BC).We analyzed the DNA from blood samples. Mutations were screened by conformation sensitive gel electrophoresis (CSGE). The amplification products displaying an abnormal pattern were sequenced using automatic system. The amplification of BCR-ABL rearrangement was studied in interphase nuclei using Vysis extra-signaling probe. Real time quantitative PCR (RQ-PCR) of BCR-ABL transcripts was performed in a subset of 44 patients to assess molecular response, using Light Cycler (Roche®), Syber Green Method.Results: Eleven mutations from 82 evaluable patients were detected (13%). Seven were in p- loop: M244R (1), L247A (1), G250E (2), Q252H (1), E255K (2), and four in the Imatinib binding: T315I (3), H318L (1). Two patients had BCR-ABL amplifications with 4–6 signals in interphase nucleous and one of them showed extramedullary involvement in skin nodules. Five patients had clonal evolution with double Ph chromosome. Most of mutations were found in patients in late chronic phase (6/11: 54%). The detection occurred at a median of 49 months (range 12–154) after diagnosis. Eighty four percent of cases studied with RQ-PCR, had null molecular response (<1Log Reduction). Imatinib dose in patients with mutations had been escalated to 600–800 mg/day previously.Conclusion: This is the first multicentric study in spanish-speaking South America. T315I was the most frequent mutation detected in our study and was associated with poor outcome. We found 13% of resistant cases with point mutations, all of them located in the p-loop or imatinib binding.This is an ongoing study and further recruitment is needed to confirm these findings. Early detection of mutations can have prognostic implication and allow therapeutic intervention such as dose escalation, combination therapy or second generation tyrosine kinase inhibitors.

Two Mutations (1717 T〉C, GP IX;124del145, GPIb beta) Occur Frequently among Patients with Bernard Soulier Syndrome in India.

Bernard Soulier syndrome (BSS) is a rare (1:1000000) hereditary bleeding disorder of platelet adhesion, caused by a qualitative or quantitative defect of glycoprotein (GP)Ib/IX/V complex. We report here the molecular basis of BSS in eight unrelated patients from India. The diagnosis was based on low platelet count, presence of giant platelets and aggregometry studies (absence of response to ristocetin) in eight unrelated patients who presented with mucocutaneous bleeding at 17.9 years of age (range:9 months–60yrs). Flow cytometry to assess surface GPIb/IX/V complex using anti CD42b antibodies showed reduced (7.7%–21.1%, n=6) expression. Genomic DNA was screened for mutations in the GPIb alpha, GPIb beta, GP IX genes by a novel PCR and conformation sensitive gel electrophoresis method. Mutations were identified in all the 8 patients. Six different mutations, including missense (n=4) mutations, a deletion and a duplication were identified of which 5 are novel. Three novel missense mutations were identified in a patient (CD42b:21.1%) of which, Tyr492His in GPIb alpha gene was in heterozygosity and the Glu129His, Leu132Pro mutations in GPIb beta was detected in a double homozygous condition. All these substitutions occur at a highly conserved codon in the transmembrane region of GPIb alpha and beta chains, a region critical for anchoring the glycoprotein subunits into the platelet membrane. A novel homozygous deletion of 22 bp in exon 2 of GPIb beta (124del145, p.Arg17CysfsX14) gene was identified in 3 different patients (CD42b: range, 10.6–15.9%). This deletion occurs at a sequence flanking the leucine rich repeat at the NH2-side of GPIb beta protein results in a premature termination of translation (PTC) and a truncated mutant with 30 amino acids. A novel homozygous duplication of 38 nucleotides (2119_2120 dup38) in the transmembrane region resulted in a frameshift (142AlafsX76) in one of the patient. This frameshift predicts termination after the addition of 75 amino acids. A previously reported homozygous transition (1717 T >C) in the GP IX gene resulting in a Cys8Arg substitution was identified in the remaining 3 patients (CD42b:10.02%, 7.7%). The frequent presence of GP IX 1717 T>C and GPIb beta 124del145 in 6 out of 8 (75%) patients studied, suggests that this could be due to a founder effect. This data adds significantly to the mutation database of this condition and will be useful for genetic diagnosis of this condition in India.

Identification of a common polymorphism in the TopBP1 gene associated with hereditary susceptibility to breast and ovarian cancer

Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast and/or ovarian cancer. TopBP1 (topoisomerase IIβ binding protein 1) displays sequence homology as well as functional similarities with BRCA1, and the two proteins have been suggested to function partly in the same cellular processes. TopBP1 is crucial for DNA damage and replication checkpoint controls. Based on its biological significance, we reasoned that TopBP1 is a plausible susceptibility gene for hereditary breast and/or ovarian cancer and therefore screened affected index cases from 125 Finnish cancer families for germline changes by conformation sensitive gel electrophoresis (CSGE). Altogether 19 different sequence alterations were detected. A novel heterozygous Arg309Cys variant was observed at elevated frequency in the familial cancer cases compared to healthy controls (15.2% versus 7.0%; P = 0.002). Current results suggest that Arg309Cys is a commonly occurring germline alteration possibly associated with a slightly increased breast and/or ovarian cancer risk. This is the first study reporting mutation screening of the TopBP1 gene in a familial cancer material.

Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies

Background: BARD1 was originally identified as a BRCA1-interacting protein but has also been described in tumour-suppressive functions independent of BRCA1. Several studies have indicated that the BARD1 gene is a...

Migrainous Vertigo: Mutation Analysis of the Candidate Genes CACNA1A, ATP1A2, SCN1A, and CACNB4

Migrainous vertigo (MV) is increasingly recognized as a common cause of episodic vertigo. MV displays several clinical similarities with familial hemiplegic migraine (FHM) and episodic ataxia type 2 (EA-2), which have been linked to mutations in 3 genes, CACNA1A, encoding a neuronal calcium channel alpha subunit, ATP1A2, encoding a catalytic subunit of a Na(+)/K(+)-ATPase, and most recently the voltage-gated sodium channel SCN1A. The present study explored the hypothesis that mutations in CACNA1A, ATP1A2, SCN1A, and the calcium channel beta(4) subunit CACNB4 confer susceptibility to MV. Mutation analysis of the coding exons and exon/intron junctions of CACNA1A, ATP1A2, SCN1A, and CACNB4 was performed in 14 unrelated MV patients by conformation sensitive gel electrophoresis and automated sequence analysis. Analysis of the 4 candidate genes in the 14 MV patients resulted in the identification of a total of 26 sequence variants. The silent substitution D29D in CACNB4 was observed in 2 MV patients and was not present in 46 ethnically matched control DNA samples. The remaining variants were also observed in control DNA samples and the allele frequencies of variants that resulted in amino acid substitutions were not significantly different between patients and controls. Based on this group of patients there is no evidence that the genes causing FHM and EA-2 represent major susceptibility loci for MV.

Mitochondrial DNA sequence variation and mutation rate in patients with CADASIL

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is clinically characterised by recurrent ischemic strokes, migraine with aura, psychiatric symptoms, cognitive decline and dementia. We have previously described a patient with CADASIL caused by a R133C mutation in the NOTCH3 gene and with a concomitant myopathy caused by a 5650G>A mutation in the MTTA gene in mitochondrial DNA (mtDNA). We assume that the co-occurrence of the two mutations is not coincidental and that mutations in the NOTCH3 gene may predispose the mtDNA to mutations. We therefore examined the nucleotide variation in the mtDNA coding region sequences in 20 CADASIL pedigrees with 77 affected patients by conformation-sensitive gel electrophoresis and sequencing. The sequence variation in mtDNA was then compared with that among 192 healthy Finns. A total of 180 mtDNA coding region sequence differences were found relative to the revised Cambridge reference sequence, including five novel synonymous substitutions, two novel nonsynonymous substitutions and one novel tRNA substitution. We found that maternal relatives in two pedigrees differed from each other in their mtDNA. Furthermore, the average number of pairwise differences in sequences from the 41 unrelated maternal lineages with CADASIL was higher than that expected among haplogroup-matched controls. The numbers of polymorphic sites and polymorphisms that were present in only one sequence were also higher among the CADASIL sequences than among the control sequences. Our results show that mtDNA sequence variation is increased within CADASIL pedigrees. These findings suggest a relationship between NOTCH3 and mtDNA.

Clinical and biological implication of defective p53 pathway in multiple myeloma (MM)

17516 Background: The p53 tumor suppressor is commonly inactivated by mutations. Even in tumors without mutations, there are defects in the response to p53 activation. In MM, the prognostic significance of p53 mutation is unknown, while there has been no systematic study of p53 function. We seek to address these issues in this study. Methods: p53 mutation was studied by conformation sensitive gel electrophoresis with primers encompassing exons 1 to 10 followed by sequencing of DNA fragments with altered electrophoretic pattern in newly diagnosed MM patients entered into ECOG E9486 trial where patients were randomized to receive variations of melphalan-based conventional chemotherapy (VBMCP). Fisher’s exact tests were used to compare variables between patients. Kaplan-Meier survival curves were compared using the log-rank test. To investigate p53 function, we analyzed the expression of p53, and 3 of its transcriptional targets, APAF1, p21 and MDM2, in a separate cohort of 15 normal plasma cells (PC), 14 MGUS, 13 smoldering myeloma (SMM) and 101 MM (73 new and 28 relapsed) from the Mayo Clinic who had gene expression profiling performed on the Affymetrix U133A chip (Santa Clara, Ca). Results: Two hundred and sixty-eight patients had enough materials for study and were included in the analysis. The prevalence of p53 mutation was 3% (n = 9). Five of the 9 patients (56%, p = 0.001) with mutations also had p53 deletion (studied by fluorescent in-situ hybridization) resulting in bi-allelic loss of p53. Soft tissue plasmacytomas (37% v 7%, p = 0.018), and among the common translocations, t(4;14) and t(14;16) (50% v 18%) were more common in patients with p53 mutations. Despite similar response to treatment, those with p53 mutation had very short OS (16.7 v 41.4 months, p &lt; 0.001). There was induction of p53 expression in MGUS and SMM with concurrent induction of APAF1, p21 and MDM2 whereas loss of this pattern was frequent in MM (45% in new MM and 60% in relapse MM compared to 15% in MGUS/SMM (p = 0.03)). Conclusions: p53 mutations are relatively rare in newly diagnosed MM patients but portend a short survival. However, functional abnormalities of p53 are prevalent and may be important in progression from MGUS/SMM to MM. [Table: see text]

High proportion of novel mutations of BRCA1 and BRCA2 in breast/ovarian cancer patients from Castilla-León (central Spain)

A total of 264 unrelated breast/ovarian cancer patients and 45 healthy individuals with familial antecedents referred for genetic testing were scanned for germ-line mutations in BRCA1 and BRCA2 by conformation-sensitive gel electrophoresis (CSGE) and heteroduplex analysis by capillary array electrophoresis (HA-CAE). We detected 101 distinct mutations (41 in BRCA1 and 60 in BRCA2); ten of them have not been previously reported. These mutations were c.2411_2429dup19, c.2802_2805delCAAA and c.5294A>G (p.E1725E) of BRCA1; and c.667C>T (p.Q147X), c.2683C>T (p.Q819X), c.5344_5347delAATA, c.5578_5579delAA;insT, c.8260_8261insGA, c.744+14C>T and c.8099A>G (p.Y2624C) of BRCA2. Twenty-four different mutations, including seven of the new mutations (five frameshift and two nonsense), were classified as pathogenic. These 24 alterations were found in 39 families (12.6% of all families). A remarkable proportion of deleterious mutations were found in BRCA2: 25 families carried a mutation in BRCA2 (BRCA2+; 64.1%) compared with 14 families BRCA1+ (35.9%). The highest incidences of deleterious mutations were found in families with three or more cases of site-specific breast cancer (BC) (27.4%) and families with BC and ovarian cancer (22.2%). Finally, four recurrent mutations, 3036_3039delACAA, c.5374_5377delTATG of BRCA2, as well as c.5272-1G>A and c.5242C>A (p.A1708E) of BRCA1, accounted for 44% of all of the deleterious mutations.

Recombinant Expression and Biochemical Characterization of the Unique Elongating β-Ketoacyl-Acyl Carrier Protein Synthase Involved in Fatty Acid Biosynthesis of Plasmodium falciparum Using Natural and Artificial Substrates

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.

BRCA1 and BRCA2 mutations in a South American population

A sample of 64 high-risk breast and/or ovarian cancer families from Chile were screened for germline mutations in the coding sequences and exon–intron boundaries of BRCA1 (MIN no. 113705) and BRCA2 (MIN no. 600185) genes using conformation-sensitive gel electrophoresis, and the mutations found were confirmed with direct sequencing. Seven families (10.9%) were found to carry BRCA1 mutations and three families (4.7%) had BRCA2 mutations. Six different pathogenic mutations were detected in BRCA1, four that had been reported previously (c.187_188delAG; c.300T →G, c.3450_3453delCAAG and IVS17-1G →A) and two novel mutations (c.2605_2606delTT and c.4185_4188delCAAG). In BRCA2, we found three different pathogenic mutations, two previously described (c.6174delT and c.6503_6504delTT) and one novel mutation (c.5667delT). We also identified nine variants of unknown significance (five in BRCA1 and four in BRCA2). These findings indicate that the Chilean population has a heterogeneous spectrum of prevalent BRCA mutations. Given the results obtained in our study, the screening of the entire BRCA1 and BRCA2 coding regions is necessary for the molecular genetic testing of Chilean high-risk breast/ovarian cancer patients. To our knowledge, this is the first genetic study of BRCA gene mutations conducted in Chile. The Chilean population has a well-known admixed Amerindian-Caucasian ratio and, therefore, our findings are not only important per se, but they constitute the basis for improved and more specific genetic counselling, as well as to support for preventive campaigns geared toward the Chilean population.

Outcome in acute lymphoblastic leukemia: Influence of thiopurine methyltransferase genetic polymorphisms

Current treatment protocols for childhood acute lymphoblastic leukemia always include thiopurine drugs such as 6-mercaptopurine (6-MP), which is administrated daily during maintenance therapy. Once 6-MP is an inactive prodrug, it needs to be activated to thioguanine nucleotides (TGNs) to exert cytotoxicity. However, 6-MP can also be methylated by the polymorphic enzyme thiopurine S-methyltransferase (TPMT), thus reducing TGNs formation. In order to assess the influence of TPMT polymorphism in ALL, we have used PCR/HCSGE (Horizontal Conformation-Sensitive Gel Electrophoresis) based methods to characterise molecularly a sample of 110 children with ALL. Four distinct alleles associated with TPMT deficiency (TPMT*3A, *3C, *2 and *8) were found in heterozygous individuals representing 11.8% of the sample. We have also compared several parameters related with ALL outcome between subsamples of children homozygous or heterozygous for TPMT. In the heterozygous group, 6-MP dosages were lower and the number of whole interruptions during treatment or interruptions due to toxicity as well as the number of relapses was higher compared to the other group. Despite our results do not reach statistical significance, this study emphasizes the importance to introduce the prospective analysis of TPMT genotype, prior to any ALL treatment.

Mutation analysis of SDHB and SDHC: novel germline mutations in sporadic head and neck paraganglioma and familial paraganglioma and/or pheochromocytoma

BackgroundGermline mutations of the SDHD, SDHB and SDHC genes, encoding three of the four subunits of succinate dehydrogenase, are a major cause of hereditary paraganglioma and pheochromocytoma, and demonstrate that these genes are classic tumor suppressors. Succinate dehydrogenase is a heterotetrameric protein complex and a component of both the Krebs cycle and the mitochondrial respiratory chain (succinate:ubiquinone oxidoreductase or complex II).MethodsUsing conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing to analyse genomic DNA from peripheral blood lymphocytes, here we describe the mutation analysis of the SDHB and SDHC genes in 37 patients with sporadic (i.e. no known family history) head and neck paraganglioma and five pheochromocytoma and/or paraganglioma families.ResultsTwo sporadic patients were found to have a SDHB splice site mutation in intron 4, c.423+1G>A, which produces a mis-spliced transcript with a 54 nucleotide deletion, resulting in an 18 amino acid in-frame deletion. A third patient was found to carry the c.214C>T (p.Arg72Cys) missense mutation in exon 4 of SDHC, which is situated in a highly conserved protein motif that constitutes the quinone-binding site of the succinate: ubiquinone oxidoreductase (SQR) complex in E. coli. Together with our previous results, we found 27 germline mutations of SDH genes in 95 cases (28%) of sporadic head and neck paraganglioma. In addition all index patients of five families showing hereditary pheochromocytoma-paraganglioma were found to carry germline mutations of SDHB: four of which were novel, c.343C>T (p.Arg115X), c.141G>A (p.Trp47X), c.281G>A (p.Arg94Lys), and c.653G>C (p.Trp218Ser), and one reported previously, c.136C>T, p.Arg46X.ConclusionIn conclusion, these data indicate that germline mutations of SDHB and SDHC play a minor role in sporadic head and neck paraganglioma and further underline the importance of germline SDHB mutations in cases of familial pheochromocytoma-paraganglioma.

Microdeletions involving theSCN1A gene may be common inSCN1A-mutation-negative SMEI patients

Severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome is a rare epilepsy syndrome. In 30 to 70% of SMEI patients, truncating and missense mutations in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A) have been identified. The majority of patients have truncating mutations that are predicted to be loss-of-function alleles. Because mutation detection studies use PCR-based sequencing or conformation sensitive gel electrophoresis (CSGE), microdeletions, which are also predicted to be loss-of-function alleles, can easily escape detection. We selected 11 SMEI patients with or without additional features who had no SCN1A mutation detectable with sequencing analysis. In addition, none of the patients was heterozygous for any of the SNPs in SCN1A, indicating that they were either homozygous for all SNPs or hemizygous due to a microdeletion of the gene. We subsequently analyzed these patients for the presence of microdeletions in SCN1A using a quantitative PCR method named multiplex amplicon quantification (MAQ), and observed three patients missing one copy of the SCN1A gene. All three microdeletions were confirmed by fluorescence in situ hybridization (FISH). These findings demonstrate that a substantial percentage of SCN1A-mutation-negative SMEI patients with or without additional features carry a chromosomal microdeletion comprising the SCN1A gene and that haploinsufficiency of the SCN1A gene is a cause of SMEI.

F8 Gene Mutations and ITT Response in the Italian Cohort of Haemophilia A Patients with Inhibitors.

Introduction: Immune tolerance therapy (ITT), consists of administration of daily high doses of factor VIII concentrates by intravenous infusions. It can induce tolerance to the exogenous protein and eradicate the antibody response to FVIII, which still represents the main complication of replacement therapy in haemophilia A (HA) severe cases.Methods: We have investigated 71 HA patients with inhibitors (sixty-nine severe, one moderate and one mild cases). We screened the patients for the causative mutations in the F8 gene using Long Range PCR for the intron22 inversion, multiplex-PCR for intron1 inversion or conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing for other mutation types.Results: Diverse genetic defects were detected in the severe cases, with a predominance of gross mutations: F8 gene inversions, large deletions and nonsense mutations account for 68% of the mutations, whereas in the two non severe patients specific missense mutations were identified. ITT has been attempted in 16 HR patients of this cohort but failed in 5 cases.Conclusion: We confirmed that the presence of inhibitors correlates well with the presence of null mutations, as reported by Schwaab et al (Thromb Haemost 1995). Several predictive factors for ITT outcome have been so far taken into consideration but no correlation has been made between F8 gene defect and ITT response. In our cohort 2 large deletions, 2 intron22 inversions and 1 nonsense mutations failed to respond. For the remaining 2 large deletions no ITT were attempted so no information are available. Large deletions appear to be a high risk genetic factor both for inhibitor development and for long term inhibitor persistence and ITT unresponsiveness. The identification of these patients at very high risk of inhibitor development and ITT unresponsiveness by mutation analysis should therefore be strongly recommended soon after the diagnosis, even because of the high cost of the therapy.

Functional Analysis of p53 in the JMW Myeloma Cell Line.

Background: The inactivation of p53 by deletion of 17p13 is observed in 10% of multiple myeloma patients and is associated with shorter survival. In this study we were interested in assessing p53 function in the JMW myeloma cell line established from a patient with aggressive disease and circulating plasma cells. We characterized the plasma cells derived from the patient with the disease from the initial stage of procurement ofpatient tumor cells to permanence as a cell line.Results: At diagnosis, 95% of the cells had monosomy 17 and 4% of the cells had a FISH pattern consistent with interstitial deletion of p53 (17p13). However, we observed clonal evolution in that at 3 months from diagnosis, 70% of the patient cells exhibited a deletion of the p53 locus while 28% of the cells had a monosomy 17. This may be attributed to favored clonal expansion of the cells with the deletion of p53 (17p13). To resolve this issue we performed further interphase and metaphase FISH and found an unbalanced complex translocation resulting in LOH of 13q and 17p (der13 t(13;17)(q?;p?). In addition, we screened the remaining p53 gene for mutations by conformation-sensitive gel electrophoresis (CSGE). PCR fragments showing abnormal CSGE-banding patterns (in exons 5 and 8) were further characterized by DNA sequence analysis and we identified a G to A splice site mutation generating an alternate splice site 21 bp into exon 5. This mutation results in a mutant protein lacking amino acids 126–132 in the DNA binding domain of p53. We observed no differences in the expression level of p53 mRNA as determined by RT-PCR analysis. We determined the effect of the deletion on p53 protein expression and function. We examined the effect of DNA damage induced by UV radiation on p53 protein levels in JMW cells, using the myeloma cell line H929 (expressing wildtype p53) as a positive control for expression. The JMW cells were exposed to UV radiation (20 J/m2) and the cells were harvested for western blot analysis 5 hours after UV damage. An increase in the level of p53 protein was observed in JMW as well as the H929 cells following UV treatment. We then investigated the activation of p53 downstream targets. The CDK inhibitor p21WAF1/CIP1 is a well-characterized p53 target that mediates G1 cell cycle arrest. Thus, we examined the levels of p21 protein following UV treatment of JMW and H929 cells. Strikingly, JMW cells lacked p21 expression in untreated cells as well as UV exposed cells suggesting that the characterized deletion in the DNA binding domain is an inactivating mutation.Conclusion: Abrogation of p53 function was observed in the JMW cells. Further analysis of the p53 pathway in JMW and other myeloma cell lines will provide an in depth understanding of the role of p53 in disease progression. The study of the p53 pathway in myeloma is highly relevant, as we have shown it to be associated with relapsing and aggressive disease, particularly with extramedullary myeloma. Our studies suggest that malignant plasma cells have the ability to survive outside of the bone marrow microenvironment but a p53 dependent checkpoint results in cell death. In clones with p53 defects the cells survive and result in extramedullary disease, including plasma cell leukemia.

Plasma Cells from Secondary Plasma Cell Leukemia Display Different Cytogenetics Pattern When Compared with Primary Plasma Cell Leukemia.

Background: Plasma cell leukemia (PCL) is a rare plasma cell (PC) dyscrasia which may be designated as primary (PPCL) or de novo PCL when recognized at the time of diagnosis and secondary (SPCL) when there is leukemic transformation of a previously recognized multiple myeloma (MM). PCL has been reported to exhibit distinct clinical and immunophenotypic features that distinguish it from MM, but little is known about the specific genetics of this disease. To better understand the genetic features of the clonal PC from PPCL and SCPL we assessed in these patients the molecular and cytogenetic abnormalities most commonly found in MM.Patients and Methods: In our study we analyzed 3 groups of patients; 18 with PPCL, 23 with SPCL and a control group of 345 newly diagnosed MM previously published. Diagnostic criteria for PCL followed the presence of >2 x103/L PC in PB (Kyle et al. Arch Intern Med 1974; 133–813–8). Cytogenetic abnormalities were assessed by the c-Ig FISH method; mutational analysis was performed using Conformational-sensitive gel electrophoresis (CSGE). Methylation was analyzed by methyl specific PCR (MSP) after bisulfite genomic DNA modification.Results: Translocation of immunoglobulin heavy chain with Cyclin D1 (t (11; 14)) was present in 76.9% (10 of 13) of PPCL, 71% (5 of 7) of SPCL, in contrast to 15.8% (53 of 336) of MM cases. Translocations' involving the FGFR3-MMSET genes (4p16.3 locus) and c-MAF (16q32) genes were not observed among 10 patients studied with PPCL, but were present in 12.7 % each in SPCL. The t (4;14)(p16;q32) was present in 12.7% cases of MM and the t(14;16)(q32;q23) was present in 4.6 % of MM cases. Deletion of 17p13.1 (p53 locus) was found in 10% (37 of 345) of MM cases, 53.8% (7 of 13) for PPCL and 42.8% (3 of 7) for SPCL. Deletion of 13q was present in 54.2% (176 of 325) of MM, 76.9 % (10 of 13) of PPCL and 57.1% of SPCL. Ras mutations were found in 15% (2/13) of PPCL, located in codon 1 and 2 of the K-ras gene. In addition, 23% for SPCL presented mutations in the N-ras gene. Two mutations were located in codon 2 of N-ras and 1 patients in showed a mutation in codon 1 of K-ras gene. Ras mutations were present in 27% of MM cases. Mutations for p53 gene were present in 30.7% (4/13) of PPCL, 17% (3/17) for SPCL and 5% for MM. Methylation specific PCR for p16 gene was found in 23% (3/13) of PPCL, 29% of SPCL and 33.9% (149/439) of MM. Hypermethylation of p14 was not detected in PPCL but a 23 % was found in SPCL.Conclusions: In this study we demonstrate that PCs from PPCL more frequently carry the t (11; 14). Comparing SPCL and MM, PC's from de novo PPCL do not show t (4; 14) or (16; 14). Deletion of 13q was more prevalent among patients with PPCL. Ras mutations appear to be as frequent in MM as in PPCL and SPCL. Mutations in p53 appeared to be more prevalent in PPCL than in SPCL and MM. Hypermethylation of p16 does not differ while hypermethylation of p14 is more frequent in SPCL than PPCL. These characteristics may lead to a different onset or disease evolution of PPCL compared to MM and secondary plasma cell leukemia and this may be important for diagnostic and treatment.

Mutational analysis of the rhodopsin gene in Chinese ADRP families by conformation sensitive gel electrophoresis

Retinitis pigmentosa is a very heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasian population. To investigate the frequency and pattern of RHO point mutations in Chinese patients with ADRP, we have screened the five coding exons and splice sites of the RHO gene in 50 unrelated probands from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified each in one family, thus the frequency of RHO mutations among ADRP families in this study is less than 14% (2 / 50 = 4%, 95% confidence interval: 1–14%), lower than that in Europe and North America, which may reflect an ethnic difference between Chinese and Caucasian populations. Loss of all phosphorylation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del). CSGE was found to be a sensitive, simple and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.

Translational Research: Basic Mechanisms

Abstract 1 Arthur J. Barela, 2 Salina P. Waddy, 1 Jay G. Lickfett, 3 Jessica Hunter, 3 Aimee Anido, 2 Sandra L. Helmers, 1 Alan L. Goldin, and 3 Andrew Escayg ( 1 Microbiology & Molecular Genetics, University of California, Irvine, CA ; 2 Neurology, Emory University, Atlanta, GA ; and 3 Human Genetics, Emory University, Atlanta, GA ) Rationale: Generalized epilepsy with febrile seizures plus (GEFS+) is a familial seizure disorder that is characterized by febrile seizures persisting beyond six years of age and a variety of afebrile epilepsy subtypes. To date, mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, SCN1B, and the GABAA receptor subunit GABRG2 have been identified in GEFS+ families. We identified a 33-member, 4-generation family with a clinical presentation consistent with GEFS+ from the Emory University Epilepsy Clinic. Methods: The coding exons of SCN1A, SCN2A, SCN1B and GABRG2 were PCR amplified from genomic DNA of the affected proband. The PCR products were screened by conformation sensitive gel electrophoresis and interesting variants were tested by direct sequencing of independent PCR products. The identified SCN1A mutation was constructed in a cDNA clone encoding the orthologous rat Nav1.1 sodium channel and the electrophysiological properties were characterized in the absence and presence of the β1 subunit in Xenopus oocytes. A computational model was used to analyze how the effects of the mutation might alter action potential generation in a neuron. Results: The nucleotide substitution C2575T was identified in exon 14 of SCN1A. Co-segregation of this mutation was observed in five additional affected members. No mutations were observed in SCN2A, SCN1B and GABRG2. The substitution neutralized a positively charged arginine by replacement with cysteine (R859C) in the S4 voltage sensor of domain II. R859 is evolutionarily conserved in all mammalian sodium channels as well as in sodium channels from lower organisms. The R859C mutation caused a ∼6 mV positive shift in the voltage-dependence of activation in both the absence and presence β1. The mutation did not alter the voltage-dependence of inactivation, nor did it affect the kinetics of fast inactivation or recovery from fast inactivation. Mutant channels entered into the slow inactivated state at a comparable rate to that of wild-type channels, but recovery from slow inactivation was significantly slower than for wild-type channels. Computational analysis suggests that neurons expressing the mutant channels have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Conclusions: The R859C mutation alters two aspects of sodium channel function, both of which should lead to decreased neuronal excitability. This result is in contrast to most previous sodium channel mutations that cause GEFS+, which have changes that are predicted to increase neuronal firing. (Supported by grants from NIH (NS48336, ALG), McKnight Foundation (34653, ALG), CURE (AE), March of Dimes (5-FY02-250, AE) and Emory University Research Council (2002120, AE).)

PCR products with heterozygous mutations containing two types of heteroduplexes

To confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes. An insertion-deletion mutation in the exon 1 of KRT9 gene (497delAinsGGCT), which caused Chinese epidermolytic palmoplantar keratoderma (EPPK) was investigated by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and denaturing high-performance liquid chromatography(DHPLC). Two heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected. PCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes. It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), conformation-sensitive gel electrophoresis (CSGE), DHPLC and heteroduplex analysis (HA), etc.