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A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.
We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initiation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.
We present a case of beta-thalassemia intermedia involving a 13-year-old boy of Northern European descent. His mother, father and older sister have normal hematologic indices. Molecular studies demonstrate that the proband carries a novel mutation of the beta-globin gene initiation codon (ATG-->AAG) which should give rise to beta(0)-thalassemia trait. The possibility of non-paternity was excluded, indicating that the novel mutation was the result of a de novo event. A review of the literature indicates that mutations involving the beta-globin gene initiation codon can give rise to a more severe phenotype than is generally associated with most other beta(+) or beta(0) mutations.
In the present study we investigate the impact of highly stable coding region secondary structures on mRNA translation efficiency. By introducing antisense segments into the 3'non-translated region of human alpha-globin mRNA we are able to synthesize a series of transcripts in which site-specific secondary structures are introduced without altering the primary structure of the 5' non-translated region, the coding region, or the encoded protein product. Coding region duplexes in close proximity to the AUG initiation codon are found to inhibit translation severely to a degree equal to that of a duplex that extends into the 5' non-translated region. In contrast, mRNAs containing duplexes positioned further 3' in the coding region translate at levels that are significantly higher although are still below those of native alpha-globin mRNA. The primary determinant of translation inhibition by coding region duplexes appears to be the proximity of the duplex to the AUG initiation codon and reflects a parallel inhibition of monosome formation. These data demonstrate that extensive coding region secondary structures suppress translation to a minimal or to a substantial degree depending on their distance from the initiation codon.
Three French families with triose phosphate isomerase (TPI) deficiency were studied, and 2 new mutations giving rise to null alleles were observed: a frameshift mutation with deletion of the 86-87 TG dinucleotide in codon 29 (TPI Alfortville) and a T-->A transversion in nucleotide 2 of the initiation codon (TPI Paris). The first mutation occurred in compound heterozygosity with the frequent E105D mutation. The second mutation occurred in association with the 2-nucleotide promoter variant (-43G,-46A). In a third family, the propositus was an E105D homozygote. In the TPI Paris family, the coinheritance of the -43,-46 promoter variant appeared to exert little, if any, effect on TPI enzyme activity, a finding consistent with 2 previous reports that questioned the putative role of the promoter polymorphism as a true deficiency variant. Similarly, the further coinheritance of glucose-6-phosphate dehydrogenase (G6PD) A- (202 G-->A/376 A-->G) appeared to have little effect on the observed phenotype. Compound heterozygosity for the E105D mutation with the null allele TPI Alfortville appeared to lead to a more severe clinical syndrome than did E105D homozygosity, suggesting that compound heterozygosity with null alleles may lead to more profound clinical abnormalities than homozygosity with missense alleles. A simple, rapid polymerase chain reaction and restriction enzyme procedure for the E105D mutation was developed for prenatal diagnosis in one family and subsequently used for screening in the other families.
An initiation codon mutation ATG-->ATA of the beta-globin gene was found in seven members of three generations of a family living in northern Sweden. This mutation, which has not previously been described, changes the initiation codon for methionine into a codon for isoleucine and will then result in a beta zero-thalassemic phenotype. The affected family members all present hematological findings typical for beta-thalassemic trait, with slight anemia, marked microcytosis, and increased levels of Hb A2.
Promoters active in the germline produce OVO-A and OVO-B mRNAs encoding isoforms of a putative transcription factor. The isoforms have a common C2H2 zinc-finger domain but different N-termini that include potential effector domains. Single point mutations in three dominant-negative ovoD mutations result in new in-frame initiation codons in OVO-B mRNAs and amino acid substitutions within charged regions of OVO-A proteins. Three lines of evidence suggest that the dominant activity is due to the new initiation codons in OVO-B mRNAs and not the amino acid substitutions in OVO-A. First, we made a fourth ovoD allele by inserting a new in-frame AUG. This ovoD4 allele encodes a nearly full-length OVO-A isoform from OVO-B mRNAs. Second, engineered stop codons in ovoD1 downstream of the new AUG abolished dominant negative activity. Third, a substantial deletion of an OVO-A region encoding a highly charged amino acid domain fully rescued loss-of-function ovo alleles. These data suggest that ovoD mutations result in inappropriate expression of OVO-A in the female germline.
Tooth development is under strict genetic control. Oligodontia is defined as the congenital absence of 6 or more permanent teeth, excluding the third molar. The occurrence of non-syndromic oligodontia is poorly understood, but in recent years several cases have been described where a single gene mutation is associated with oligodontia. Several studies have shown that MSX1 and PAX9 play a role in early tooth development. We screened one family with non-syndromic oligodontia for mutations in MSX1 and PAX9. The pedigree showed an autosomal-dominant pattern of inheritance. Direct sequencing and restriction enzyme analysis revealed a novel heterozygous A to G transition mutation in the AUG initiation codon of PAX9 in exon 1 in the affected members of the family. This is the first mutation found in the initiation codon of PAX9, and we suggest that it causes haploinsufficiency.
A polymorphism at the first of two potential translation initiation codons in the vitamin D receptor (VDR) gene defined by the FokI restriction endonuclease has been associated with reduced bone mineral density (BMD) among Caucasian, Asian, and Mexican-American women. We tested the hypothesis that the FokI polymorphism is related to markers of osteoporotic risk in 104 community-dwelling African-American women aged 65 years and older. Six percent of the African-American women had the ff genotype, 32% were heterozygous, and 63% had the FF genotype. FokI genotype frequencies did not differ from Hardy-Weinberg expectations. Hip and calcaneal BMD, calcaneal ultrasound attenuation and hip geometry from pelvic radiographs did not differ significantly by FokI genotypes or between women with and without the rare FokI allele. There was also no association between the FokI polymorphism and biochemical markers of bone turnover or fractional calcium absorption. We conclude that the VDR start codon polymorphism does not have a major influence on osteoporotic risk in older African-American women.
A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.
The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.
Statistical analysis of the AUG initiation codon context in several plant organisms identified a nucleotide preference in some positions around the AUG. Sixteen AUG contexts were studied using transient expression in tobacco, maize and Norway spruce. Besides the importance of A or G at position -3, we revealed the role of positions -2, -1 for which AA or CC were found to be the best for tobacco and maize, respectively. GC (positions +4, +5) were also found to be important in both tobacco and maize. Finally, we identified a variation in context efficiency according to cell type, since A was better than G at position -3 in tobacco leaf protoplasts, while both nucleotides were equally efficient in tobacco suspension cells.
Sunflower mitochondrial DNA contains a single copy of the cob gene. The gene begins with the unusual GTG initiation codon and lies in a transcription unit having a different organization with respect to that common to the other six known sequences from plant species which include both monocotyledons and dicotyledons.
More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT1151, results in Lesch-Nyhan syndrome (LNS), and the other, HPRTIllinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRTIllinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRTIllinois. These data provide support for the hypothesis that patient RT, or variant HPRTIllinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.
To determine whether sequence context influences the ability of an AUG triplet to be recognized as an initiator codon by eukaryotic ribosomes, single nucleotide changes were introduced near the translational start site in a cloned preproinsulin gene. Maximum synthesis of preproinsulin occurred when a purine, preferably adenosine, was located three nucleotides upstream from the initiator codon. Adenosine is found most frequently in that position in natural mRNAs.
In this report, we studied on a homoplasmic T12338C change in mitochondrial DNA (mtDNA), which substituted methionine in the translational initiation codon of the NADH dehydrogenase subunit 5 gene ( ND5) with threonine. This nucleotide change was originally identified in two mtDNAs belonging to haplogroup F2 by our previous complete sequencing of 48 mtDNAs. Since then, a total of 76 F2 mtDNAs have been identified by the variations occurring in the hypervariable segments and coding regions among more than 3,000 individuals across China. As the T12338C change was detected in 32 samples representing various sub-clades of the F2 haplogroup while not in 14 non-F2 controls, we believe that the T12338C change is specific to the F2 haplogroup. As F2 and its sub-clades were widely distributed in normal individuals of various Chinese populations, we conclude that T12338C is not pathogenic. In addition, based on the average distribution frequency, haplotype diversity and nucleotide diversity of haplogroup F2 in the populations across China, the T12338C nucleotide substitution seems to have been occurred in north China about 42,000 years ago. Our results provided a good paradigm for distinguishing a polymorphic change from a pathogenic mutation based on mtDNA phylogeny.
Murine leukemia virus (MuLV) codes for two precursors of the group-specific antigens, Pr65gag and Pr75gag, in vivo. While Pr65gag is the precursor to the virion structural proteins, Pr75gag undergoes glycosylation and is found on the surface of the infected cell as gp85gag, and it is thought to play a role in virus maturation and spread. Pr65gag synthesis starts at an AUG codon within a favourable initiation context (AAUAUGG at positions 618 to 624). The gp85gag start codon is upstream but its precise location is not known. To map the initiation codon of gp85gag, we used deletion and site-directed mutagenesis of the leader sequence of MuLV RNA and in vitro translation of the RNAs. Synthesis of the MuLV gp85gag protein appears to be initiated at a CUG codon located within a favourable context (ACCCUGG at positions 354 to 359 for Moloney-MuLV). The possible function of gp85gag was investigated by expressing Moloney-MuLV and Friend-MuLV proviral DNA and mutants deficient for gp85gag synthesis in mouse and rat cells. The results indicate that the gp85gag protein probably facilitates the spread of virus infection in tissue culture.
To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.