Deletion Of Codon Research Articles

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a −127 G→A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.

Development of new cytomegalovirus UL97 and DNA polymerase mutations conferring drug resistance after valganciclovir therapy in allogeneic stem cell recipients

Background We report on two allogeneic stem cell transplant recipients who developed cytomegalovirus disease associated with new viral mutations that conferred antiviral drug resistance. Methods Blood specimens obtained during symptomatic disease were analyzed for mutations in the CMV UL97 and DNA polymerase genes and new mutations were assessed by recombinant phenotyping. Results Rising cytomegalovirus (CMV) antigenemia occurred after 4–5 months of preemptive valganciclovir therapy, followed by symptomatic CMV disease including fatal pneumonia in one case. In one case, a new viral UL97 mutation (deletion of codons 601–603) was found which conferred 15-fold increased ganciclovir resistance. In the other case, a known UL97 resistance mutation M460V and a new DNA polymerase ( pol) mutation D413A were found. D413A conferred ganciclovir and cidofovir resistance. Conclusions Known and newly discovered drug resistance mutations arising during preemptive therapy may complicate post-transplant CMV disease in stem cell recipients. Improved recombinant phenotyping methods enable the rapid quantitation of the resistance conferred by newly identified UL97 and pol mutations.

Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania -Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum N -Acetylglucosamine-1-Phosphate Transferase

A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.

Novel Mechanism of β0 Thalassemia: Missense Mutation of the Last Nucleotide of β Globin Exon 1 (G->C) Leads to Undetectable Transcript and Mutant Peptide.

Hemoglobin Monroe (Hb Monroe) results from a point mutation (G->C) in the last nucleotide of β globin exon 1, which is also the penultimate nucleotide of codon 30 of the β globin mRNA (AGG->ACG/Arg->Thr). Hb Monroe was described seventeen years ago simultaneously by two groups: in an African American female with β thalassemia intermedia, wherein the thalassemia was thought to result from highly unstable peptide (Hemoglobin. 1989; 13:67); and in a North African female with compound β thalassemia (Proc Natl Acad Sci U S A. 1989; 86:1041) wherein the Hb Monroe mutation was thought to result in abnormal pre-mRNA splicing as detected in an in vitro cell-free transcription assay. We evaluated a 31-year old previously asymptomatic woman of Asian Indian (Bengali) descent, who presented with flu like symptoms and found to have low hemoglobin level (9.5 gm/dL), microcytosis (MCV 68 fL), moderately elevated liver enzymes and serum ferritin concentration of 3000 ng/ml. A liver biopsy revealed increased liver iron and significant fibrosis. Hemoglobin analysis, which was interpreted as compound heterozygosity for HbE/β0 thalassemia, revealed HbF: 51%, HbE: 43.2%, HbA2: 5.8%. β globin gene sequencing showed Hb Monroe and E mutations. The asymptomatic brother of the proband had borderline anemia (Hb 12 gm %), microcytosis (MCV 70 fl), HbF: 8.5 %, HbA: 87.2%, HbA2: 5.0% and was heterozygous for Hb Monroe mutation by Bme 15801 restriction enzyme analysis of genomic DNA. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe mutation and whether this mutation in our subjects is present on African haplotype or had arisen independently. Since we could not detect the mutant peptide either in fresh hemolysate or reticulocyte enriched preparations; we expanded the peripheral blood erythroid progenitor cells in vitro of both proband and her brother. Hemoglobin analysis by both HPLC and mass spectrophotometry did not detect Hb Monroe peptide in the expanded cells. β globin cDNA from the reticulocytes and expanded erythroid progenitors was amplified using three different primer sets and no splice variants were detectable. Sequencing of the amplified cDNA revealed only normal β globin mRNA transcript. Other β globin gene mutations cis to Hb Monroe are being ruled out; to date, we have not found any promoter region or stop codon mutations, deletions or splicing mutations from promoter - 90 region to 3′ UTR including poly-A region. Haplotype analysis revealed a different haplotype from the two African American patients, indicating an independent origin of Hb Monroe mutation in our cases. Interestingly, both of our cases have elevated HbF, as did the two originally reported Hb Monroe patients (16.5 and 85%) and a currently unreported African American patient with sickle cell - β0 thalassemia due to Hb Monroe (11.4%). Hb F was also high in a group of nine patients reported with sickle cell - β0 thalassemia due to Hb Monroe (3.1%– 8.9%; Hemoglobin. 1998; 22:153). We conclude that this missense mutation, IVS1-1 (G->C/Arg 30 Thr) results in undetectable transcript and mutant Hb peptide leading to β0 thalassemia. The molecular mechanism of the undetectable mutant transcript is being investigated.

Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling.

Autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) colocalize in the apical monocilia of renal epithelial cells. Mouse and human renal cells without PC1 protein show impaired ciliary mechanosensation, and this impairment has been proposed to promote cystogenesis. However, most cyst epithelia of human ADPKD kidneys appear to express full-length PC1 and PC2 in normal or increased abundance. We show that confluent primary ADPKD cyst cells with the novel PC1 mutation DeltaL2433 and with normal abundance of PC1 and PC2 polypeptides lack ciliary PC1 and often lack ciliary PC2, whereas PC1 and PC2 are both present in cilia of confluent normal human kidney (NK) epithelial cells in primary culture. Confluent NK cells respond to shear stress with transient increases in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), dependent on both extracellular Ca(2+) and release from intracellular stores. In contrast, ADPKD cyst cells lack flow-sensitive [Ca(2+)](i) signaling and exhibit reduced endoplasmic reticulum Ca(2+) stores and store-depletion-operated Ca(2+) entry but retain near-normal [Ca(2+)](i) responses to ANG II and to vasopressin. Expression of wild-type and mutant CD16.7-PKD1(115-226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independent ciliary localization signal. However, the coiled-coil domain is required for CD16.7-PKD1(115-226) expression to accelerate decay of the flow-induced Ca(2+) signal in NK cells. These data provide evidence for ciliary dysfunction and polycystin mislocalization in human ADPKD cells with normal levels of PC1.

Epidermal growth factor receptor mutations and susceptibility to targeted therapy in lung cancer

According to 2002 estimates, 1.35 million people were diagnosed with and 1.18 million died of lung cancer worldwide. Recently, a new class of medications targeting signal transduction pathways has come into focus in the treatment of various malignancies. In lung cancer, the molecules gefitinib and erlotinib which target the intracellular kinase domain of the epidermal growth factor receptor (EGFR), cause significant tumour responses and, in the case of erlotinib, a survival benefit in patients with previously treated cancers. Responses were most pronounced in female non-smokers with adenocarcinoma histology. These patients were found more likely to harbour mutations of the receptor kinase domain, including in-frame deletions in exon 19 (such as deletions of codons 746-750) and point deletions in exon 21 (such as L858R). Other EGFR kinase domain mutations have been found to confer resistance (T790M) or differential susceptibility to erlotinib and gefitinib (E884K). Gene amplification of EGFR also may predict sensitivity, although the mechanism by which this occurs is unclear, because level of expression detected by immunohistochemistry has not been correlated with increased sensitivity. Phenotypic and genotypic epithelial to mesenchymal transition may be an indicator of resistance to EGFR kinase inhibitors. In this article, we review efforts that have been undertaken to identify genomic determinants of drug susceptibility to EGFR tyrosine kinase inhibitors, with particular focus on the role of gene mutations.

Chikungunya Virus Strains, Reunion Island Outbreak

Chikungunya Virus Strains, Reunion Island Outbreak

A codon deletion confers resistance to herbicides inhibiting protoporphyrinogen oxidase

Herbicides that act by inhibiting protoporphyrinogen oxidase (PPO) are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites. Evaluation of a PPO-inhibitor-resistant A. tuberculatus biotype revealed that resistance was a (incompletely) dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare.

Familial gastrointestinal stromal tumor with homo-/hemizygous kit exon 11 deletion: Genotypic, histopathologic, radiographic, and therapeutic findings

9527 Background: Germ-line mutations in the kit receptor tyrosine kinase gene have been described in families with a propensity to develop gastrointestinal stromal tumor (GIST). We describe a large kindred with multiple individuals with GIST and describe the genotype, clinical presentation, radiographic imaging, surgical findings, imatinib-sensitivity, and histopathologic appearance. Patients and Methods: A large kindred with multiple GISTs were identified after the proband patient was referred to our institution. The history of each affected and unaffected family member was obtained in person or via telephone. A medical questionnaire was sent to family members and appropriate medical records, tumor tissue and radiographic imaging was obtained. Histopathologic specimens from four of the family members with GIST was obtained, the diagnosis confirmed and three specimens were used to sequence kit exon 9, 11, 13, 15 and 17. Results: Fifteen members were identified in this family with histopathologic diagnosis of GIST or probable GIST. Surgical findings revealed multiple tumors arising from the submucosa of the small intestine. Histopathology revealed microscopic proliferation of the myenteric plexus with areas of microscopic tumor nodularity. Four patients in this kindred were treated with imatinib and had radiographic and clinical evidence of therapeutic benefit. A deletion of codon 579 in exon 11 of the kit gene was identified in tumor and normal tissue of this family. Evaluation of one GIST revealed loss of heterozygosity in kit exon 11 suggesting a homozygous mutation or loss of the wild-type locus. Conclusion: This study describes a kindred with a propensity to develop GIST in association with hyperpigmentation and melanocytic nevi, but not dysphagia. The germline deletion of codon 579 appears to confer proliferation and nodularity of the myenteric plexus, synchronous tumors at presentation, a metastatic phenotype, and in vivo sensitivivity to imatinib therapy. No significant financial relationships to disclose.

GLUTATHIONES-TRANSFERASE OMEGA 1 AND OMEGA 2 PHARMACOGENOMICS

Glutathione S-transferase omega 1 and omega 2 (GSTO1 and GSTO2) catalyze monomethyl arsenate reduction, the rate-limiting reaction in arsenic biotransformation. As a step toward pharmacogenomic studies of these phase II enzymes, we resequenced human GSTO1 and GSTO2 using DNA samples from four ethnic groups. We identified 31 and 66 polymorphisms in GSTO1 and GSTO2, respectively, with four nonsynonymous-coding single nucleotide polymorphisms (cSNPs) in each gene. There were striking variations among ethnic groups in polymorphism frequencies and types. Expression constructs were created for all eight nonsynonymous cSNPs, as well as a deletion of codon 155 in GSTO1, and those constructs were used to transfect COS-1 cells. Quantitative Western blot analysis, after correction for transfection efficiency, showed a reduction in protein level of greater than 50% for the GSTO1 Tyr32 variant allozyme compared with wild type (WT), whereas levels for the Asp140, Lys208, Val236, and codon 155 deletion variant constructs were similar to that of the WT. For GSTO2, the Tyr130 and Ile158 variant allozymes showed 50 and 84% reductions in levels of expression, respectively, compared with WT, whereas the Ile41 and Asp142 allozymes displayed levels similar to that of WT GSTO2. Rabbit reticulocyte lysate degradation studies showed that the GSTO1 Tyr32 and the GSTO2 Tyr130, Ile158, and Asp142/Ile158 variant allozymes were degraded more rapidly than were their respective WT allozymes. These observations raise the possibility of functionally significant pharmacogenomic variation in the expression and function of GSTO1 and GSTO2.

Effects of Mutations and Truncations on the Kinetic Behavior of IIAGlc, a Phosphocarrier and Regulatory Protein of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

IIAGlc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate --> Enzyme I --> HPr --> IIAGlc --> IICBGlc --> Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Delta7 and Delta16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain "docks" IIAGlc to the lipid bilayer of membranes containing IICBGlc.

Lack of association with TorsinA haplotype in German patients with sporadic dystonia

Genetic variability at loci underlying autosomal dominant forms of neurologic diseases may also contribute to the risk of developing sporadic disease. The most common cause of autosomal dominantly inherited, primary generalized dystonia is a deletion of a glutamate codon (GAG) in the TorsinA gene on chromosome 9q34.1 Sporadic (typically focal) dystonia is considerably more common. A strong association of sporadic dystonia with the TorsinA haplotype T2 was recently reported in the Icelandic population.2 The use of population isolates in genetic studies is increasingly popular. However, genetic and environmental homogeneity does not reflect the diversity of humankind as a whole. Furthermore, genetic drift influences the genotypic makeup of population isolates. Thus, findings derived from studies of population isolates need to be replicated in normal, out-bred populations to confirm their relevance outside the population isolate.3 We explored whether sporadic dystonia is also associated with any of the TorsinA haplotypes in a more typical, nonisolated population. We assessed TorsinA haplotypes …

HOXA1 mutations are not a common cause of Duane anomaly

HOXA1 mutations are not a common cause of Duane anomaly

Emergence of a Three Codon Deletion ingagp17 in HIV Type 1 Subtype C Long-Term Survivors, and General Population Spread

In a population-based study in northern Malawi we investigated HIV-1 subtype C gag and env gene sequences associated with long-term survival. DNA samples were available from 31 individuals surviving between population surveys carried out in the 1980s and 1990s. Most survivors with paired sequences dating from the 1980s and the 1990s had a three codon deletion in the gag p17 region of the sequence retrieved from the sample collected in the 1990s that was not present in the sequence from the same individual dating from the 1980s. This deletion was also not present in any other 1980s sequences from Malawi, but was common in samples collected in Malawi in the 1990s. The deletion is equivalent to the loss of three amino acids in the D helix region of the gag protein, and may be associated with longer survival and onward transmission.

Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires

It has been suggested that codon insertion and/or deletion may represent a mechanism that, along with hypermutation, contributes to the affinity maturation of antibodies. We used repertoire cloning to examine human antibodies directed against 3 carbohydrate antigens and 1 protein antigen for the presence of such modifications. We find that both the insertion and deletion of codons occur frequently in antigen-specific responses following vaccination. Codon insertions and deletions were observed most often in the complementarity determining regions, and less frequently in the framework regions, of VH, Vκ, and Vλ gene segments, and involved motifs known to be preferred targets of somatic hypermutation. Clonal lineage analysis shows that these events occur through out the course of the somatic maturation of individual antibody clones. We also determined that these alterations of paratope structure have varying effects on the relative affinity of the binding site for its cognate antigen.ReviewersThis article was reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein.Open peer reviewReviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein. For the full reviews, please go to the Reviewers' comments section.

Two New Human DMT1 Mutations in a Compound Heterozygous Patient with Microcytic Anemia and Low Iron Stores.

DMT1 is a divalent metal transporter with 12 transmembrane domains. It is expressed at the apical membrane of duodenal enterocytes, where it mediates pH-dependent uptake of Fe2+. In erythroid cells, it is found in the endosomal membrane where it transfers iron internalized through the transferrin-transferrin receptor pathway from the endosome to the cytosol. The same homozygous G>A substitution resulting in the G185A replacement is responsible for a severe hypochromic microcytic anemia in both the mk mouse and in the Belgrade rat. In humans, a homozygous G>C mutation has been described in a Czech patient, affecting the last nucleotide of exon 12. This mutation leads to the G399A replacement, without affecting the transport function of the protein. However, this mutation also induces a preferential in-frame skipping of exon 12, albeit not in all tissues. Accordingly, the patient has impaired iron acquisition in erythrocytes while duodenal iron absorption is increased leading to progressive iron overload.Here, we report a female patient born in 1996, with low birth weight and hypochromic microcytic anemia (Hb = 7.5 g/dl; MCV = 53 fL). She was transfused at day 0 and put on oral iron treatment. She was then lost to follow-up for five years. At the age of five, more extensive explorations showed a persistent microcytic anemia. The bone marrow displayed normal cellularity, 30% of nucleated cells were erythroid precursors with a moderate maturation defect, acidophilic forms being under-represented as compared to more immature forms. Soluble transferrin receptors were increased (8.3 mg/L; N = 0.83–1.76). Following oral iron therapy, serum ferritin levels remained low (15–25 μg/L; N = 14–197) despite an increase in transferrin saturation from 68 to 95 %. This high transferrin saturation resulted from the combination of reduced transferrin levels (1.64 g/L, N = 2.2–4.0) and increased serum iron levels (35 μmol/L; N = 11–24). On the other hand, hemoglobin raised from 7 to 9 g/dL only through increased number of RBC (5 to 5.7 T/L), since MCV and MCHC remained unchanged.We sequenced the entire transferrin receptor cDNA in this patient and found no mutation. We then sequenced the exons and the intron-exon boundaries of the DMT1 gene and found two heterozygous mutations. One mutation was a deletion of a GTG codon in exon 5, leading to the V114 in-frame deletion, in transmembrane domain 2. The other mutation is a G>T substitution in exon 8 leading to the G212V replacement in transmembrane domain 5. Both parents were asymptomatic, the father being heterozygous for the delV114 mutation and the mother heterozygous for the G212V mutation.This is the second patient described with a neonatal hypochromic microcytic anemia due to DMT1 mutations. Our data suggest that the two combined DMT1 mutations are responsible for the defect in iron utilization by erythroid cells, resulting in persistant microcytosis and impaired red cell maturation. The effect of the mutations on intestinal iron absorption is more difficult to evaluate since iron therapy allowed serum iron and transferrin saturation to increase but serum ferritin remained low and hemoglobin did not reach normal values.

Mutational Analysis of the c-KIT AND PDGFR?? in a Series of Molecularly Well-Characterized Synovial Sarcomas

The c-KIT and the platelet-derived growth factor receptor alpha (PDGFRalpha) have been shown to be important for tumor growth and progression in several soft-tissue sarcomas, including synovial sarcomas (SSs). It has been suggested that these c-KIT-positive cases might benefit from a tyrosine kinase inhibitor therapy. In this study, we analyze a series of SSs to investigate the presence of c-KIT and PDGFRalpha mutations with the aim of selecting those for a more adequate and appropriate therapy. We analyzed fresh-frozen tissues from 12 SSs (8 primary tumors and 4 nude mice xenotransplants from primary tumors). RNA was extracted to identify the presence of the SYT-SSX gene fusion to confirm the SS diagnosis. Mutational analysis of exons 9, 11, 13, and 17 of c-KIT and exons 12 and 18 of PDGFRalpha was performed by direct sequencing. Immunohistochemical analysis of c-KIT, PDGFRalpha, and p-PDGFRalpha was also performed. All analyzed cases showed the presence of SYT-SSX gene fusion transcripts confirming the diagnosis of SS, 10 carried the SYT-SSX1 fusion, and 2 the SYT-SSX2. Immunohistochemical analysis showed expression of c-KIT in 3 cases in which no molecular alterations were detected. For the PDGFRalpha, we observed an in-frame deletion of codons 554 and 555 in a case which also showed a strong immunopositivity for the phosphorylated form of PDGFRalpha. PDGFRalpha expression was observed in 8 cases. We suggest that a more exhaustive mutational analysis of the c-KIT and PDGFRalpha genes should be performed to ascertain which cases would really benefit from a tyrosine kinase inhibitor therapy in SS.

Novel Germline Mutation of KIT Associated With Familial Gastrointestinal Stromal Tumors and Mastocytosis

Gastrointestinal stromal tumors (GISTs) are often associated with activating KIT mutations, affecting regulatory domains of the KIT tyrosine kinase. Sporadic mastocytosis in adults is usually also caused by KIT mutations that, however, activate KIT by affecting the intracellular enzymatic site of the molecule. Most GISTs respond to KIT inhibitors that bind to the enzymatic site; in most cases of mastocytosis, however, the modified enzymatic site is not affected by these drugs. We present a kindred with both familial GISTs and mastocytosis that express a novel germline KIT mutation in exon 8, resulting in deletion of codon 419 and affecting the extracellular domain of KIT. This mutation activates KIT, and the mutant KIT is inhibited by the tyrosine kinase inhibitor imatinib mesylate. Our studies identify a new regulatory region in the KIT molecule and strongly suggest that patients with extracellular KIT mutations respond to tyrosine kinase inhibitors.

Functional Significance and Clinical Phenotype of Nontruncating Mismatch Repair Variants of

Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes.

Early Selection of a New UL97 Mutant with a Severe Defect of Ganciclovir Phosphorylation after Valaciclovir Prophylaxis and Short-Term Ganciclovir Therapy in a Renal Transplant Recipient

We describe the emergence of a new ganciclovir resistance mutation in the UL97 gene of human cytomegalovirus, deletion of codon 601, after valaciclovir and short-term ganciclovir therapy following kidney transplantation. Its role in ganciclovir resistance was supported by decreased ganciclovir phosphorylation in a recombinant vaccinia virus system.