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PubMed2006

Homozygous nonsense mutation in the FOXE3 gene as a cause of congenital primary aphakia in humans.

Congenital primary aphakia (CPA) is a rare developmental disorder characterized by the absence of lens, the development of which is normally induced during the 4th-5th wk of human embryogenesis. This original failure leads, in turn, to complete aplasia of the anterior segment of the eye, which is the diagnostic histological criterion for CPA. So far, the genetic basis for this human condition has remained unclear. Here, we present the analysis of a consanguineous family with three siblings who had bilateral aphakia, microphthalmia, and complete agenesis of the ocular anterior segment. We show that a null mutation in the FOXE3 gene segregates and, in the homozygous state, produces the mutant phenotype in this family. Therefore, this study identifies--to our knowledge, for the first time--a causative gene for CPA in humans. Furthermore, it indicates a possible critical role for FOXE3 very early in the lens developmental program, perhaps earlier than any role recognized elsewhere for this gene.

Adult↗
PubMed2001

Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.

Affinity maturation of the humoral immune response is caused by single base changes that are introduced into the V regions of the Ig genes during a brief period of B cell differentiation. It has recently become possible to study V region mutation in some human Burkitt's lymphoma cell lines that mutate their V regions and express surface markers that suggest they arose from the malignant transformation of germinal center B cells. Ramos Burkitt's cells constitutively mutate their V regions at a rate of approximately 2 x 10(-5) mutations/bp/generation. However, the sequencing of unselected V regions suggested that our Ramos cell line was progressively losing its ability to undergo V region hypermutation. To accurately quantify this process, subclones with different nonsense mutations in the mu heavy chain V region were identified. Reversion analysis and sequencing of unselected V regions were used to examine the clonal stability of V region hypermutation. Even after only 1 month in culture, stable and unstable subclones could be identified. The identification of mutating and non-mutating subclones of Ramos provided a unique opportunity to identify factors involved in the mutational process. Differential gene expression between mutating and non-mutating Ramos clones was examined by RT-PCR and cDNA microarray analyses. We found that the expression of activation-induced cytidine deaminase (AID), a putative cytidine deaminase, correlated with mutation rates in Ramos subclones. These results suggest that the hypermutation phenotype is inherently unstable in Ramos and that long culture periods favor outgrowth of non-mutating cells that express lower levels of AID.

Burkitt Lymphoma↗
PubMed2004

A TP53-truncating germline mutation (E287X) in a family with characteristics of both hereditary diffuse gastric cancer and Li-Fraumeni syndrome.

Mutations in CDH1, which encodes E-cadherin, have been associated with hereditary diffuse gastric cancer (HDGC) in Western populations but have not been shown to play a major role in Asians. Recently, a patient with familial gastric cancer (FGC) was shown to harbor a germline mutation in the TP53 gene, which encodes p53 and has been previously associated with Li-Fraumeni Syndrome (LFS). To determine whether mutations in TP53 are associated with FGC in Asians, we screened the entire coding region of TP53 in probands from 23 Korean FGC families. We identified a nonsense (E287X) TP53 germline mutation in a family whose history is compatible with both HDGC and LFS. Two members of this family (SNU-G2) were afflicted with brain tumors, seven with gastric cancers, two with sarcomas, and one with both gastric cancer and a sarcoma. The E287X TP53 mutation segregated with the cancer phenotype in the family members from whom DNA samples were available. To our knowledge, this is the first report of a large family with both HDGC and LFS. Our results suggest that TP53 mutational screening in FGC families should be interpreted with caution because additional TP53 mutation-carrying HDGC families may also show LFS-related phenotypes.

Base Sequence↗
PubMed2005

SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway.

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.

14-3-3 Proteins↗
PubMed2004

Chimeric RNA/ethylene-bridged nucleic acids promote dystrophin expression in myocytes of duchenne muscular dystrophy by inducing skipping of the nonsense mutation-encoding exon.

Editing of dystrophin mRNA by induction of exon skipping, using antisense oligonucleotides, has been proposed as one way to generate dystrophin expression in Duchenne muscular dystrophy (DMD) patients. Here, antisense chimeric oligonucleotides consisting of RNA and a new modified nucleic acid are tested for activity to induce skipping of an exon containing a nonsense mutation. In a Japanese DMD case, a nonsense mutation (R1967X) due to a single nucleotide change in exon 41 of the dystrophin gene (C5899T) was identified. Oligonucleotides consisting of 2'-O-methyl RNA and a new 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) were designed to bind the mutation site of exon 41, and their ability to induce exon 41 skipping in dystrophin mRNA was evaluated. Finally, among the specific oligonucleotides tested, an 18-mer RNA/ENA chimera was found to have the strongest activity, inducing exon 41 skipping in nearly 90% of dystrophin mRNA. Accordingly, nearly 90% of cultured myocytes were shown to be dystrophin positive by immunohistochemical analysis. Western blot analysis disclosed the presence of nearly normal-sized dystrophin up to 1 week after the transfection. Our results suggest that an RNA/ENA chimera can be used to express dystrophin in DMD.

Base Sequence↗
PubMed2001

Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans.

Mutations in the smu-1 gene of Caenorhabditis elegans were previously shown to suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. We show that a presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. We have used reverse transcription-PCR and RNase protection to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. We have identified smu-1 molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. We propose that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes.

Alternative Splicing↗
PubMed2003

Novel types of mutation in the choroideremia ( CHM) gene: a full-length L1 insertion and an intronic mutation activating a cryptic exon.

Choroideremia (CHM) is a progressive chorioretinal degeneration caused by mutations in the widely expressed CHM gene on chromosome Xq21. The product of this gene, Rab escort protein (REP)-1, is involved in the posttranslational lipid modification and subsequent membrane targeting of Rab proteins, small GTPases that play a key role in intracellular trafficking. We have searched for mutations of the CHM gene in patients with choroideremia by analysis of individual CHM exons and adjacent intronic sequences PCR-amplified from genomic DNA and by reverse transcription (RT)-PCR analysis of the coding region of the CHM mRNA. In 35 patients, at least 21 different causative CHM defects were identified. These included two partial CHM gene deletions and an insertion of a full-length L1 retrotransposon into the coding region of the CHM gene, a type of mutation that has not been previously reported as a cause of CHM. We also detected nine different nonsense mutations, five of which are recurrent, a small deletion, a small insertion, and at least five distinct splice site mutations, one of which has been described previously. Moreover, we report for the first time the identification of an intronic mutation remote from the exon-intron junctions that creates a strong acceptor splice site and leads to the inclusion of a cryptic exon into the CHM mRNA. Finally, in an affected male who did not have a mutation in any of the CHM exons or their splice sites, the deletion of a complete exon from the CHM mRNA was observed.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Molecular defects in type 3 von Willebrand disease: updated results from 40 multiethnic patients.

Type 3 von Willebrand disease (VWD) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe hemorrhagic symptoms. We have characterized at the molecular level a group of 40 patients (12 Italians, 14 Iranians, and 14 Indians) to evaluate genetic heterogeneity among these populations. Some of these patients have been previously investigated by us (mutations shown in italics); they are included in this study to provide a more comprehensive pattern of gene defects in type 3 VWD. Patients' DNA were first tested for more frequently reported mutations, then screened by single-strand conformation polymorphism and direct sequence analysis. Fifty gene defects were identified, of which 45 are novel. As expected most of these defects caused null alleles, 17 being nonsense mutations (Q218X, W222X, R365X, R373X, Y610X, W642X, E644X, Q706X, Q1311X, S1338X, Q1346X, Y1542X, R1659X, E1981X, E2129X, R2434X, and Q2544X), 12 small deletions (191delG, 276delT, 788del24, 2016del4, 2157delA, 2269delCT, 2435delC, 4092delAC, 6182delT, 7294delGT, 7683delT, and 8241del9), 4 small insertions (4414insC, 7130insC, 7137insT, and 7674insC), 8 possible splice site mutations (1110(-1)G-->A, 1946(-4)C-->T, 3108(+5)G-->A, 3379(+1)G-->A, 5053(+1)G-->A, 5170(+10)C-->T, 6977(-1)G-->C, and 7729(+7)C-->T), 8 candidate missense mutations (D47H, S85P, D141N, D141Y, C275S, C1071F, C2174G, and C2804Y), and 1 large gene deletion (exons 23-52). Only 2 of these patients have developed alloantibodies against VWF. This study extend our previous finding that mutations responsible for type 3 VWD are scattered throughout the entire VWF gene and that there is no founder effect in these three populations studied.

Codon, Nonsense↗
PubMed2005

NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome.

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.

Child↗
PubMed2002

Genetics of familial episodic vertigo and ataxia.

The familial episodic ataxias are prototypical inherited channelopathies that result in episodes of vertigo and ataxia triggered by stress and exercise. Episodic ataxia type 1 (EA-1) is caused by missense mutations in the potassium channel gene KCNA1, whereas episodic ataxia type 2 (EA-2) is caused by missense and nonsense mutations in the calcium channel gene CACNA1A. These ion channels are crucial for both central and peripheral neurotransmission. Within the last few years, the genetic mechanisms underlying these relatively rare familial episodic ataxia syndromes have been worked out. They provide a model for understanding the mechanisms of more common recurrent vertigo and ataxia syndromes, particularly those associated with migraine. Migraine affects as many as 15-20% of the general population, and it has been estimated that about 25% of patients with migraine experience spontaneous attacks of vertigo and ataxia. We identified 24 families with migraine and benign recurrent vertigo inherited in an autosomal dominant fashion. These families have numerous features in common with EA-1 and EA-2 (particularly EA-2), suggesting that benign recurrent vertigo may be an inherited channelopathy. An ion channel mutation shared by brain and inner ear could explain the combined central and peripheral features of the syndrome.

Calcium Channels↗
PubMedDate not supplied

Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.

Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology.

ATP-Binding Cassette Transporters↗
PubMed1999

Germline mutations in the multiple endocrine neoplasia type 1 gene: evidence for frequent splicing defects.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome characterized by parathyroid hyperplasia, pituitary adenomas, and neuroendocrine tumors of the pancreas and duodenum. In 1997, the MEN1 tumor suppressor gene was identified, and numerous germline mutations have been reported to be distributed throughout the gene. We used single strand conformational variant (SSCV) analysis to search for germline mutations in the members of 33 kindreds with a confirmed diagnosis of MEN 1. SSCV analysis revealed 25 conformational variants representing germline mutations that are predicted to result in loss of normal menin function. Twenty different disease-associated mutations were identified: five resulting in potential abnormal RNA splicing, two missense mutations, seven nonsense mutations, and six frameshift mutations. The aberrant splice products were identified and confirmed by RT-PCR and direct sequence analysis for two of the five splice mutations. Sixteen of the 20 (80%) mutations identified have not been previously reported. Mutations were not identified in eight kindreds with signs and symptoms consistent with MEN 1. The SSCV analysis revealed mutations in 76% (25 of 33) of the kindreds investigated, thus showing SSCV analysis to be a reliable mutation detection strategy. One-fifth of the mutations identified in this study involve intron sequences, therefore, highlighting the importance of including intron sequences in the search for germline mutations in the MEN1 gene. The need to investigate the entire gene when characterizing new MEN 1 families presents challenges in the translation of genetic studies to efficient clinical diagnostic tests.

Chromosomes, Human, Pair 11↗
PubMed2004

Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase.

We identified an autosomal recessive infantile-onset symptomatic epilepsy syndrome associated with developmental stagnation and blindness. Assuming a founder effect in a large Old Order Amish pedigree, we carried out a genome-wide screen for linkage and identified a single region of homozygosity on chromosome 2p12-p11.2 spanning 5.1 cM (maximum lod score of 6.84). We sequenced genes in the region and identified a nonsense mutation in SIAT9, which is predicted to result in the premature termination of the GM3 synthase enzyme (also called lactosylceramide alpha-2,3 sialyltransferase). GM3 synthase is a member of the sialyltransferase family and catalyzes the initial step in the biosynthesis of most complex gangliosides from lactosylceramide. Biochemical analysis of plasma glycosphingolipids confirmed that affected individuals lack GM3 synthase activity, as marked by a complete lack of GM3 ganglioside and its biosynthetic derivatives and an increase in lactosylceramide and its alternative derivatives. Although the relationship between defects in ganglioside catabolism and a range of lysosomal storage diseases is well documented, this is the first report, to our knowledge, of a disruption of ganglioside biosynthesis associated with human disease.

Blindness↗
PubMed1999

Clinical and molecular genetic analysis of 19 Wolfram syndrome kindreds demonstrating a wide spectrum of mutations in WFS1.

Wolfram syndrome is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset diabetes mellitus and progressive optic atrophy. mtDNA deletions have been described, and a gene (WFS1) recently has been identified, on chromosome 4p16, encoding a predicted 890 amino acid transmembrane protein. Direct DNA sequencing was done to screen the entire coding region of the WFS1 gene in 30 patients from 19 British kindreds with Wolfram syndrome. DNA was also screened for structural rearrangements (deletions and duplications) and point mutations in mtDNA. No pathogenic mtDNA mutations were found in our cohort. We identified 24 mutations in the WFS1 gene: 8 nonsense mutations, 8 missense mutations, 3 in-frame deletions, 1 in-frame insertion, and 4 frameshift mutations. Of these, 23 were novel mutations, and most occurred in exon 8. The majority of patients were compound heterozygotes for two mutations, and there was no common founder mutation. The data were also analyzed for genotype-phenotype relationships. Although some interesting cases were noted, consideration of the small sample size and frequency of each mutation indicated no clear-cut correlations between any of the observed mutations and disease severity. There were no obvious mutation hot spots or clusters. Hence, molecular screening for Wolfram syndrome in affected families and for Wolfram syndrome-carrier status in subjects with psychiatric disorders or diabetes mellitus will require complete analysis of exon 8 and upstream exons.

Adolescent↗
PubMed2006

Thymic carcinoid in multiple endocrine neoplasia 1: genotype-phenotype correlation and prevention.

Thymic carcinoid is a rare multiple endocrine neoplasia type 1 (MEN1)-associated tumour that is a major cause of death in MEN1 patients. Here, we describe a previously unreported MEN1 family in which two siblings presented with malignant thymic carcinoids. All six siblings share a novel nonsense mutation Q395X on exon 8 of the MEN1 gene. The index patient developed a thymic carcinoid despite an earlier prophylactic transcervical thymectomy, and one other sibling had an incidental malignant thymic carcinoid discovered following prophylactic thymectomy, both cases demonstrating the weakness and strength of this surgical approach. We then review the spectrum of germline MEN1 mutations associated with thymic carcinoids to evaluate the possibility of a genotype-phenotype correlation. In the 22 separate MEN1 families with thymic carcinoids, all but two (91%) have mutations coding for a truncated protein. There is clearly a high prevalence of truncating mutations in MEN1-related thymic carcinoids although when compared with the prevalence of truncating mutations in all reported MEN1 mutations, it is not statistically significant (P = 0.39). Further studies are warranted to evaluate pathways of tumorigenesis of thymic carcinoids with regard to loss of function of menin.

Adult↗
PubMed2006

Incidence of CSF3R mutations in severe congenital neutropenia and relevance for leukemogenesis: Results of a long-term survey.

Point mutations in the gene for the granulocyte colony-stimulating factor (G-CSF) receptor CSF3R have been implicated in the progression of severe congenital neutropenia (CN) to leukemia. In this study we present data on a total of 218 patients with chronic neutropenia, including 148 patients with CN (23/148 with secondary malignancies). We detected CSF3R nonsense mutations at 17 different nucleotide positions (thereof 10 new mutations) which lead to a loss of 1 to all 4 tyrosine residues in the intracellular domain of the receptor. Of 23 patients with CN with signs of malignant transformation, 18 (78%) were shown to harbor a CSF3R mutation, indicating that these mutations, although not a necessary condition, are highly predictive for malignant transformation even if detected in a low percentage of transcripts. In serial analyses of 50 patients with CSF3R mutations we were able to follow the clonal dynamics of mutated cells. We could demonstrate that even a highly clonal hematopoiesis did not inevitably show a rapid progression to leukemia. Our results strongly suggest that acquisition of a CSF3R mutation is an early event in leukemogenesis that has to be accompanied by cooperating molecular events, which remain to be defined.

Adolescent↗
PubMed1999

Mutations in the growth hormone releasing hormone receptor: a new form of dwarfism in humans.

We describe a recently identified new form of dwarfism due to isolated growth hormone (GH) deficiency, secondary to inactivating mutations in the GH-releasing hormone receptor (GHRHR) gene. The identical nonsense mutations in the extracellular domain of the GHRHR (E72X or E50X, depending on whether the signal peptide is included in the numbering) has been independently described in three families residing on or originating from the Indian subcontinent (Pakistan, the Bombay region, and Delft near Sri Lanka). Another inactivating mutation, involving the donor splice site of intron 1, has been identified in a population in north-eastern Brazil. Genetic transmission is autosomal recessive; the gene is located on the short arm of chromosome 7. Affected subjects have severe isolated GH deficiency and postnatal growth failure, with a mean adult height of 130 cm for men and 114 cm for women (7-8 standard deviations below the norm). Dwarfism is proportional; a characteristic feature is relative microcephaly, which results in a 'miniaturized adult', eumorphic aspect. Bone age and puberty are delayed, but fertility appears normal. This new syndrome corresponds to the human homologue of the previously identified 'little mouse'.

Adult↗
PubMed2001

In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.

Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

Animals↗