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PubMed2003

Extracellular matrix protein 1 gene (ECM1) mutations in lipoid proteinosis and genotype-phenotype correlation.

The autosomal recessive disorder lipoid proteinosis results from mutations in extracellular matrix protein 1 (ECM1), a glycoprotein expressed in several tissues (including skin) and composed of two alternatively spliced isoforms, ECM1a and ECM1b, the latter lacking exon 7 of this 10-exon gene (ECM1). To date, mutations that either affect ECM1a alone or perturb both ECM1 transcripts have been demonstrated in six cases. However, lipoid proteinosis is clinically heterogeneous with affected individuals displaying differing degrees of skin scarring and infiltration, variable signs of hoarseness and respiratory distress, and in some cases neurological abnormalities such as temporal lobe epilepsy. In this study, we sequenced ECM1 in 10 further unrelated patients with lipoid proteinosis to extend genotype-phenotype correlation and to add to the mutation database. We identified seven new homozygous nonsense or frameshift mutations: R53X (exon 3); 243delG (exon 4); 507delT (exon 6); 735delTG (exon 7); 785delA (exon 7); 892delC (exon 7) and 1190insC (exon 8), as well as two new compound heterozygous mutations: W160X/F167I (exon 6) and 542insAA/R243X (exons 6/7), none of which were found in controls. The mutation 507delT occurred in two unrelated subjects on different ECM1 haplotypes and may therefore represent a recurrent mutation in lipoid proteinosis. Taken with the previously documented mutations in ECM1, this study supports the view that exons 6 and 7 are the most common sites for ECM1 mutations in lipoid proteinosis. Clinically, it appears that mutations outside exon 7 are usually associated with a slightly more severe mucocutaneous lipoid proteinosis phenotype, but neurological features do not show any specific genotype-phenotype correlation.

Adolescent↗
PubMed1996

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.

Adult↗
PubMed2005

Mitochondrial myopathy and rhabdomyolysis associated with a novel nonsense mutation in the gene encoding cytochrome c oxidase subunit I.

Mitochondrial DNA (mtDNA) mutations associated with rhabdomyolysis are rare but have been described in sporadic cases with mutations in the cytochrome b and cytochrome c oxidase (COX) genes and in 3 cases with tRNALeu mutation. We report a novel heteroplasmic G6708A nonsense mutation in the mtDNA COI gene encoding COX subunit I in a 30-year-old woman with muscle weakness, pain, fatigue, and one episode of rhabdomyolysis. Histochemical examination of muscle biopsy specimens revealed reduced COX activity in the majority of the muscle fibers (approximately 90%) and frequent ragged red fibers. Biochemical analysis showed a marked and isolated COX deficiency. Analysis of DNA extracted from single fibers revealed higher levels of the mutation in COX-deficient fibers (> 95%) compared with COX-positive fibers (1%-80%). The mutation was not detected in a skin biopsy, cultured myoblasts, or blood leukocytes. Nor was it identified in blood leukocytes from the asymptomatic mother, indicating a de novo mutation that arose after germ layer differentiation. Western blot analysis and immunohistochemical staining revealed that reduced levels of COX subunit I were accompanied by reduced levels of other mtDNA encoded subunits, as well as nuclear DNA encoded subunit IV, supporting the concept that COX subunit I is essential for the assembly of complex IV in the respiratory chain.

Adult↗
PubMed2000

Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene.

The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.

Animals↗
PubMed2005

Fishy taint in chicken eggs is associated with a substitution within a conserved motif of the FMO3 gene.

Fishy odor of urine and other secretions is a characteristic of trimethylaminuria in humans, resulting from loss-of-function mutations in the flavin-containing mono-oxygenase isoform FMO3. A similar phenotype exists in cattle, in which a nonsense mutation in the bovine orthologue causes fishy off-flavor in cow's milk. The fishy odor is caused by an elevated level of excreted odorous trimethylamine (TMA), due to deficient oxidation of TMA. We report the mapping of a similar disorder (fishy taint of eggs) and the chicken FMO3 gene to chicken chromosome 8. The only nonsynonymous mutation identified in the chicken FMO3 gene (T329S) changes an evolutionarily highly conserved amino acid and is associated with elevated levels of TMA and fishy taint in the egg yolk in several chicken lines. No differences in the expression of FMO3 were found among individuals with different associated genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The results support the importance and function of the evolutionarily conserved motif FATGY, which has been speculated to be a substrate recognition pocket of N-hydroxylating siderophore enzymes and flavin-containing mono-oxygenases.

Alleles↗
PubMed2005

Identification of NOM1, a nucleolar, eIF4A binding protein encoded within the chromosome 7q36 breakpoint region targeted in cases of pediatric acute myeloid leukemia.

Proteins that contain the recently described MIF4G and/or MA3 domains function in translation, cell growth, proliferation, transformation, and apoptosis. Examples of MIF4G/MA3 containing proteins and their functions include eIF4G, which serves as a scaffold for assembly of factors required for translation initiation, programmed cell death protein 4 (Pdcd4) that inhibits translation and functions as a tumor suppressor, and NMD2, which is essential for nonsense-mediated mRNA decay. MIF4G and MA3 domains serve as binding sites for one or more isoforms of the eIF4A family of ATP-dependent DEAD-box RNA helicases that are required for translation and for nonsense-mediated decay. In this report, we describe the characterization of a novel MIF4G/MA3 family member called NOM1 (nucleolar protein with MIF4G domain 1) that was identified at the chromosome 7q36 breakpoint involved in 7;12 translocations associated with certain acute leukemias of childhood. NOM1, which includes a previously described EST called c7orf3, encodes a ubiquitously expressed transcript composed of 11 exons and an approximately 3 kb 3' UTR that contains several Alu repeats. The predicted NOM1 protein contains one MIF4G domain and one MA3 domain and, consistent with data obtained with other MIF4G/MA3 proteins, interacts with members of the eIF4A family of helicases. Database searches reveal that NOM1 homologs exist in several organisms and that at least two of these are essential genes. Finally, like its Saccharomyces cerevisiae homolog Sgd1p, NOM1 localizes predominantly to the nucleolus. These data demonstrate that NOM1 is a new member of the MIF4G/MA3 family of proteins and suggest that it may provide an essential function in metazoans.

Alu Elements↗
PubMedDate not supplied

Functional analysis of RUNX2 mutations in cleidocranial dysplasia: novel insights into genotype-phenotype correlations.

Cleidocranial dysplasia (CCD) is an inherited autosomal-dominant skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor, RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations alongside one polymorphism. The missense mutations were all clustered within the Runt domain and their protein products showed neither DNA binding nor transactivation. On the other hand, some mutant RUNX2 had the Runt domain intact and remained partially competent for transactivation. Coincidentally, one important phenotype of CCD, the short stature, was significantly milder in the patients with the intact Runt domain than those without. Furthermore, a remarkable correlation was found between the short stature and the number of supernumerary teeth. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed regardless of the degree of short stature or supernumerary teeth. Overall, these results suggest that CCD could result from a much smaller loss in the RUNX2 function than envisioned on the basis of the conventional haploinsufficiency model. This makes an interesting contrast to the case of familial and sporadic leukemias mediated by RUNX1 mutations, in which mutants acting in a dominant negative manner have been suggested to confer a higher propensity to develop leukemia.

Cleidocranial Dysplasia↗
PubMed2004

Telomere cap components influence the rate of senescence in telomerase-deficient yeast cells.

Cells lacking telomerase undergo senescence, a progressive reduction in cell division that involves a cell cycle delay and culminates in "crisis," a period when most cells become inviable. In telomerase-deficient Saccharomyces cerevisiae cells lacking components of the nonsense-mediated mRNA decay (NMD) pathway (Upf1,Upf2, or Upf3 proteins), senescence is delayed, with crisis occurring approximately 10 to 25 population doublings later than in Upf+ cells. Delayed senescence is seen in upfDelta cells lacking the telomerase holoenzyme components Est2p and TLC1 RNA, as well as in cells lacking the telomerase regulators Est1p and Est3p. The delay of senescence in upfDelta cells is not due to an increased rate of survivor formation. Rather, it is caused by alterations in the telomere cap, composed of Cdc13p, Stn1p, and Ten1p. In upfDelta mutants, STN1 and TEN1 levels are increased. Increasing the levels of Stn1p and Ten1p in Upf+ cells is sufficient to delay senescence. In addition, cdc13-2 mutants exhibit delayed senescence rates similar to those of upfDelta cells. Thus, changes in the telomere cap structure are sufficient to affect the rate of senescence in the absence of telomerase. Furthermore, the NMD pathway affects the rate of senescence in telomerase-deficient cells by altering the stoichiometry of telomere cap components.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Molecular, ultrastructural and functional characterization of a Spanish family with Hermansky-Pudlak syndrome: role of insC974 in platelet function and clinical relevance.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, which is genetically heterogeneous. In humans, mutations associated with this syndrome have been identified that affect four genes, most of them located in the HPS-1 gene. We evaluated the clinical, molecular, platelet ultrastructure and platelet function data obtained from one Spanish HPS patient and his relatives. The proband was compound heterozygous for a de novo nonsense mutation (Arg-131Stop), which has not been described previously, and for a common frameshift mutation (insC974). These two mutations were also identified by reverse transcription polymerase chain reaction (RT-PCR) in half the RNA, supporting the premise that they have minor effects on either transcription or RNA stability. The patient had an almost complete absence of platelet-dense granules. Accordingly, his platelets showed a small aggregatory response, reduced CD63 surface expression after platelet activation and minor serotonin uptake. Interestingly, despite the absence of clinical symptoms, two relatives carrying only one HPS-1 mutation (insC974) presented a decreased content of platelet-dense granules and showed significant reductions in platelet aggregation, expression of CD63 after platelet activation and serotonin uptake. Data show that the presence of a single mutation affecting one allele of the HPS-1 gene might have relevance in the organogenesis of platelet-dense granules, affecting platelet function. However, these functional defects were not of a great enough magnitude to have clinical significance and, thus, these subjects were clinically asymptomatic.

Blood Platelets↗
PubMed2006

Novel mutations in the OPA1 gene and associated clinical features in Japanese patients with optic atrophy.

PURPOSE: Autosomal dominant optic atrophy (ADOA) is characterized by symmetrical bilateral optic atrophy associated with reduced corrected visual acuity (VA), central or centrocecal scotoma, and color vision disturbances. The disease is genetically heterogeneous, and the OPA1 gene has been identified as the only causative gene. The aims of this study were to identify and report mutations in the OPA1 gene in Japanese patients with ADOA and to describe the clinical features associated with the mutations. DESIGN: Molecular genetic study and observational case reports. PARTICIPANTS: Nine unrelated Japanese families with optic atrophy and 8 isolated cases of optic atrophy. METHODS: Genomic DNA was extracted from peripheral leukocytes, and all exons containing the open reading frame of the OPA1 gene and the flanking intron splice sites were sequenced directly. Complete ophthalmologic examinations were performed. MAIN OUTCOME MEASURES: Direct sequencing of the OPA1 gene and clinical evaluations including VA, visual field, color vision, and disc appearance. RESULTS: Ten different heterozygous mutations, including 6 novel mutations, were detected in the OPA1 gene. The identified mutations included 5 deletions/insertions (c.2061delA, c.2098_2103delCTTAAA, c.2538insT, c.2591insC, and c.2708_2711delTTAG), 4 nonsense mutations (c.112C>T [p.R38X], c.181C>T [p.Q61X], c.946A>T [p.R316X], and c.2713C>T [p.R905X]), and 1 missense mutation (c.1635C>A [p.S545R]). The most common mutation in Caucasians (c.2708_2711delTTAG) was found in 3 unrelated families, suggesting that it is a mutational hot spot. We detected an OPA1 mutation in 8 of 9 familial cases of optic atrophy and in 4 of 8 cases that were initially considered to be sporadic from the patients' family histories. Examinations of family members of 2 sporadic probands revealed the existence of other family members with the OPA1 mutations whose phenotype was very mild or within normal limits. This indicates that patients with ADOA sometimes seem to be sporadic because of the extensive variation in the phenotype or, alternatively, a low penetrance of ADOA. CONCLUSIONS: OPA1 gene mutations are causative in most familial cases of ADOA in Japanese. Sporadic cases of optic atrophy frequently may be caused by OPA1 mutations in the Japanese population. Molecular genetic examinations are useful in determining the hereditary patterns in some cases of optic atrophy.

Adult↗
PubMed2001

Rent1, a trans-effector of nonsense-mediated mRNA decay, is essential for mammalian embryonic viability.

The ability to detect and degrade transcripts that lack full coding potential is ubiquitous but non-essential in lower eukaryotes, leaving in question the evolutionary basis for complete maintenance of this function. One hypothesis holds that nonsense-mediated RNA decay (NMD) protects the organism by preventing the translation of truncated peptides with dominant negative or deleterious gain-of-function potential. All organisms studied to date that are competent for NMD express a structural homolog of Saccharomyces cerevisiae Upf1p. We have now explored the consequences of loss of NMD function in vertebrates through targeted disruption of the Rent1 gene in murine embryonic stem cells which encodes a mammalian ortholog of Upf1p. Mice heterozygous for the targeted allele showed no apparent phenotypic abnormalities but homozygosity was never observed, demonstrating that Rent1 is essential for embryonic viability. Homozygous targeted embryos show complete loss of NMD and are viable in the pre-implantation period, but resorb shortly after implantation. Furthermore, Rent1(-/-) blastocysts isolated at 3.5 days post-coitum undergo apoptosis in culture following a brief phase of cellular expansion. These data suggest that NMD is essential for mammalian cellular viability and support a critical role for the pathway in the regulated expression of selected physiologic transcripts.

Animals↗
PubMed2003

Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene.

Mutations in the LMNA gene encoding nuclear lamins A and C are responsible for seven inherited disorders affecting specific tissues. We have analyzed skin fibroblasts from a patient with type 1B limb-girdle muscular dystrophy and from her deceased newborn grandchild carrying, respectively, a heterozygous (+/mut) and a homozygous (mut/mut) nonsense Y259X mutation. In fibroblasts(+/mut), the presence of only 50% lamins A and C promotes no detectable abnormality, whereas in fibroblasts(mut/mut) the complete absence of lamins A and C leads to abnormally shaped nuclei with lobules in which none of the analyzed nuclear proteins were detected, i.e., B-type lamins, emerin, nesprin-1alpha, LAP2beta, and Nup153. These lobules perturb cell division as fibroblast(mut/mut) cultures with large proportions of cells with dysmorphic nuclei grow more slowly than controls and the cell proliferation normalizes when the number of these abnormally shaped nuclei declines. In all fibroblasts(mut/mut), nesprin-1alpha-like emerin exhibited aberrant localization in the endoplasmic reticulum. Transfection of wild-type lamin A or C cDNAs restored the correct localization of both emerin and nesprin-1alpha. These data demonstrate that lamin C, like lamin A, interacts in vivo directly with nesprin-1alpha and with emerin and that lamin A or C is sufficient for the correct anchorage of emerin and nesprin-1alpha at the nuclear envelope in human cells.

Carrier Proteins↗
PubMed2000

Distinct mutations in the receptor tyrosine kinase gene ROR2 cause brachydactyly type B.

Brachydactyly type B (BDB) is an autosomal dominant skeletal disorder characterized by hypoplasia/aplasia of distal phalanges and nails. Recently, heterozygous mutations of the orphan receptor tyrosine kinase (TK) ROR2, located within a distinct segment directly after the TK domain, have been shown to be responsible for BDB. We report four novel mutations in ROR2 (two frameshifts, one splice mutation, and one nonsense mutation) in five families with BDB. The mutations predict truncation of the protein within two distinct regions immediately before and after the TK domain, resulting in a complete or partial loss of the intracellular portion of the protein. Patients affected with the distal mutations have a more severe phenotype than do those with the proximal mutation. Our analysis includes the first description of homozygous BDB in an individual with a 5-bp deletion proximal to the TK domain. His phenotype resembles an extreme form of brachydactyly, with extensive hypoplasia of the phalanges and metacarpals/metatarsals and absence of nails. In addition, he has vertebral anomalies, brachymelia of the arms, and a ventricular septal defect-features that are reminiscent of Robinow syndrome, which has also been shown to be caused by mutations in ROR2. The BDB phenotype, as well as the location and the nature of the BDB mutations, suggests a specific mutational effect that cannot be explained by simple haploinsufficiency and that is distinct from that in Robinow syndrome.

Amino Acid Sequence↗
PubMed2004

Hailey-Hailey disease: identification of novel mutations in ATP2C1 and effect of missense mutation A528P on protein expression levels.

ATP2C1, encoding the human secretory pathway Ca(2+)-ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey disease (HHD), an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis. In this study, we used denaturing high-performance liquid chromatography to screen all 28 exons and flanking intron boundaries of ATP2C1 for mutations in 9 HHD patients. Nine different mutations were identified. Five of these mutations, including one nonsense, one deletion, two splice-site, and one missense mutation, have not been previously reported. Recently, functional analysis of a series of site-specific mutants, designed to mimic missense mutations found in ATP2C1, uncovered specific defects in Ca(2+) and/or Mn(2+) transport and protein expression in mutant hSPCA1 polypeptides. In order to investigate the molecular and physiological basis of HHD in the patient carrying missense mutation A528P, located in the putative nucleotide binding domain of the molecule, site-directed mutagenesis was employed to introduce this mutation into the wild-type ATP2C1 (hSPCA1) sequence. Functional analyses of HHD-mutant A528P demonstrated a low level of protein expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. Analogous to conclusions drawn from our previous studies, these results further support the theory of haploinsufficiency as a prevalent mechanism for the dominant inheritance of HHD, by suggesting that the level of hSPCA1 in epidermal cells is critical.

Amino Acid Sequence↗
PubMed2007

Autosomal recessive von Willebrand disease associated with compound heterozygosity for a novel nonsense mutation (2908 del C) and the missense mutation C2362F: definite evidence for the non-penetrance of the C2362F mutation.

A novel null mutation (2908del C in exon 22) of the von Willebrand factor (VWF) gene was identified in compound heterozygosity with the missense mutation G7335T (C2362F) in exon 42 in a propositus from a new family with autosomal recessive von Willebrand disease (VWD). The propositus, referred at age 2 for severe epistaxis and prolonged bleeding after a tongue bite, had factor VIII:C 14-21 IU/dL, VWF Antigen 3-8 IU/dL and Ristocetin Cofactor activity < 3 IU/dL. Multimeric pattern showed the lack of triplet pattern and a faster mobility of central band, while heterozygotes for C2362F showed intermediate mobility compared to normal plasma and plasma from the propositus. In the propositus' family 5 subjects were heterozygotes for the C2362F mutation and 5 were heterozygotes for the cytosine deletion. Bleeding score was assessed with a detailed questionnaire in 28 heterozygotes for C2362F, 23 of whom identified in 5 previously reported families and 5 in the present one, and found to be similar to what is observed in normal controls and heterozygotes for null allele. In conclusion, the mutation C2362F is frequently observed in compound heterozygosity with null alleles in patients with recessive VWD in the Veneto region and cause bleeding only in the compound heterozygous or homozygous state.

Alleles↗
PubMed2001

Niemann-Pick disease type C: spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

Adult↗
PubMed2005

Identification of inactivating mutations in the JAK1, SYNJ2, and CLPTM1 genes in prostate cancer cells using inhibition of nonsense-mediated decay and microarray analysis.

We have developed a simple analytical method that increases the efficiency of identifying mutant genes in cell lines after the inhibition of nonsense-mediated decay (NMD). The approach assumes that the spectra of mutant genes differ between cell lines of the same tumor origin. Thus, by analyzing more than one cell line in parallel and taking into account not only changes in mRNA levels after the inhibition of NMD, but also comparing mRNA levels between cell lines before the inhibition of NMD, the vast majority of false positives were eliminated from the analysis. In this study, we used Affymetrix oligonucleotide arrays to compare mRNA profiles of two prostate cancer cell lines, PC3 and LNCaP, before and after emetine treatment. As a result of our modified approach, from the 14,500 genes present on the array, 7 were identified as candidates from LNCaP cells and 1 was identified from PC3 cells. Sequence analysis of five of these candidate genes identified gene-inactivating mutations in four of them. Homozygous mutations were found in the synaptojanin 2 (SYNJ2) and the cleft lip and palate CLPTM1 genes. Two different heterozygous mutations in the Janus kinase 1 (JAK1) gene result in complete loss of the protein in several different prostate cancer cell lines.

Codon, Nonsense↗
PubMed2002

Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L.

Mutations in the MTR gene, which encodes methionine synthase on human chromosome 1p43, result in the methylcobalamin deficiency G (cblG) disorder, which is characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia. To investigate the molecular basis of the disorder, we have characterized the structure of the MTR gene, thereby identifying exon-intron boundaries. This enabled amplification of each of the 33 exons of the gene, from genomic DNA from a panel of 21 patients with cblG. Thirteen novel mutations were identified. These included five deletions (c.12-13delGC, c.381delA, c.2101delT, c.2669-2670delTG, and c.2796-2800delAAGTC) and two nonsense mutations (R585X and E1204X) that would result in synthesis of truncated proteins that lack portions critical for enzyme function. One mutation was identified that resulted in conversion of A to C of the invariant A of the 3' splice site of intron 9. Five missense mutations (A410P, S437Y, S450H, H595P, and I804T) were identified. The latter mutations, as well as the splice-site mutation, were not detected in a panel of 50 anonymous DNA samples, suggesting that these sequence changes are not polymorphisms present in the general population. In addition, a previously described missense mutation, P1173L, was detected in 16 patients in an expanded panel of 24 patients with cblG. Analysis of haplotypes constructed using sequence polymorphisms identified within the MTR gene demonstrated that this mutation, a C-->T transition in a CpG island, has occurred on at least two separate genetic backgrounds.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗