Single Nucleotide Transversion Research Articles

Mixed insect pest populations of Diaspididae species under control of oligonucleotide insecticides: 3′-end nucleotide matters

Diaspididae are one of the most serious small herbivorous insects with piercing–sucking mouth parts and are major economic pests as they attack and destroy perennial ornamentals and food crops. Chemical control is the primary management approach for armored scale infestation. However, chemical insecticides do not possess selectivity in action and not always effective enough for the control of armored scale insects. Our previous work showed that green oligonucleotide insecticides (olinscides) are highly effective against armored and soft scale insects. Moreover, olinscides possess affordability, selectivity in action, fast biodegradability, and a low carbon footprint. Insect pest populations undergo microevolution and olinscides should take into account the problem of insecticide resistance. Using sequencing results, it was found that in the mixed populations of insect pests Dynaspidiotus britannicus Newstead and Aonidia lauri Bouche, predominates the population of A. lauri. Individuals of A. lauri comprised for 80% of individuals with the sequence 3′-ATC-GTT-GGC-AT-5′ in the 28S rRNA site, and 20% of the population comprised D. britannicus individuals with the sequence 3′-ATC-GTC-GGT-AT-5′. We created olinscides Diasp80–11 (5′-ATG-CCA-ACG-AT-3′) and Diasp20–11 (5′-ATA-CCG-ACG-AT-3′) with perfect complementarity to each of the sequences. Mortality of insects on the 14th day comprised 98.19 ± 3.12% in Diasp80–11 group, 64.66 ± 0.67% in Diasp20–11 group (p < 0.05), and 3.77 ± 0.94% in the control group. Results indicate that for maximum insecticidal effect it is necessary to use an oligonucleotide insecticide that corresponds to the dominant species. Mortality in Diasp80–11 group was accompanied with significant decrease in target 28S rRNA concentration and was 8.44 ± 0.14 and 1.72 ± 0.36 times lower in comparison with control (p < 0.05) on the 10th and 14th days, respectively. We decided to make single nucleotide substitutions in Diasp20–11 olinscide to understand which nucleotide will play the most important role in insecticidal effect. We created three sequences with single nucleotide transversion substitutions at the 5′-end – Diasp20(5′)-11 (A to T), 3′-end – Diasp20(3′)-11 (T to A), and in the middle of the sequence – Diasp20(6)-11 (6th nitrogenous base of the sequence; G to C), respectively. As a result, mortality of mixed population of the field experiment decreased and comprised 53.89 ± 7.25% in Diasp20(5′)-11 group, 40.68 ± 4.33% in Diasp20(6)-11 group, 35.74 ± 5.51% in Diasp20(3′)-11 group, and 3.77 ± 0.94% in the control group on the 14th day. Thus, complementarity of the 3′-end nucleotide to target 28S rRNA was the most important for pronounced insecticidal effect (significance of complementarity of nucleotides for insecticidal effect: 5′ nt < 6 nt < 3′ nt). As was found in our previous research works, the most important rule to obtain maximum insecticidal effect is complete complementarity to the target rRNA sequence and maximum coverage of target sequence in insect pest populations. However, in this article we also show that the complementarity of 3′-end is a second important factor for insecticidal potential of olinscides. Also in this article we propose 2-step DNA containment mechanism of action of olinscides, recruiting RNase H. The data obtained indicate the selectivity of olinscides and at the same time provide a simple and flexible platform for the creation of effective plant protection products, based on antisense DNA oligonucleotides.

Characterization of terminal flowering cowpea (Vigna unguiculata (L.) Walp.) mutants obtained by induced mutagenesis digs out the loss-of-function of phosphatidylethanolamine-binding protein

Cowpea (Vigna unguiculata (L.) Walp) is one of the major food legume crops grown extensively in arid and semi-arid regions of the world. The determinate habit of cowpea has many advantages over the indeterminate and is well adapted to modern farming systems. Mutation breeding is an active research area to develop the determinate habit of cowpea. The present study aimed to develop new determinate habit mutants with terminal flowering (TFL) in locally well-adapted genetic backgrounds. Consequently, the seeds of popular cowpea cv P152 were irradiated with doses of gamma rays (200, 250, and, 300 Gy), and the M1 populations were grown. The M2 populations were produced from the M1 progenies and selected determinate mutants (TFLCM-1 and TFLCM-2) from the M2 generation (200 Gy) were forwarded up to the M5 generation to characterize the mutants and simultaneously they were crossed with P152 to develop a MutMap population. In the M5 generation, determinate mutants (80–81 days) were characterized by evaluating the TFL growth habit, longer peduncles (30.75–31.45 cm), erect pods (160°- 200°), number of pods per cluster (4–5 nos.), and early maturity. Further, sequencing analysis of the VuTFL1 gene in the determinate mutants and MutMap population revealed a single nucleotide transversion (A-T at 1196 bp) in the fourth exon and asparagine (N) to tyrosine (Y) amino acid change at the 143rd position of phosphatidylethanolamine-binding protein (PEBP). Notably, the loss of function PEPB with a higher confidence level modification of anti-parallel beta-sheets and destabilization of the protein secondary structure was observed in the mutant lines. Quantitative real-time PCR (qRT-PCR) analysis showed that the VuTFL1 gene was downregulated at the flowering stage in TFL mutants. Collectively, the insights garnered from this study affirm the effectiveness of induced mutation in modifying the plant’s ideotype. The TFL mutants developed during this investigation have the potential to serve as a valuable resource for fostering determinate traits in future cowpea breeding programs and pave the way for mechanical harvesting.

Global high-throughput genotyping of organellar genomes reveals insights into the origin and spread of invasive starry stonewort (Nitellopsis obtusa)

Aquatic invasive species are damaging to native ecosystems. Preventing their spread and achieving comprehensive control measures requires an understanding of the genetic structure of an invasive population. Organellar genomes (plastid and mitochondrial) are useful for population level analyses of invasive plant distributions. In this study we generate complete organellar reference genomes using PacBio sequencing, then use these reference sequences for SNP calling of high-throughput, multiplexed, Illumina based organellar sequencing of fresh and historical samples from across the native and invasive range of Nitellopsis obtusa (Desv. in Loisel.) J.Groves, an invasive macroalgae. The data generated by the analytical pipeline we develop indicate introduction to North America from Western Europe. A single nucleotide transversion in the plastid genome separates a group of five samples from Michigan and Wisconsin that either resulted from introductions of two closely related genotypes or a mutation that has arisen in the invasive range. This transversion will serve as a useful tool to understand how Nitellopsis obtusa moves across the landscape. The methods and analyses described here are broadly applicable to invasive and native plant and algae species, and allow efficient genotyping of variable quality samples, including 100-year-old herbarium specimens, to determine population structure and geographic distributions.

Molecular Analysis of Ephrin A4 and Ephrin B1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.

An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T → A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing “rescue.”

A Novel Missense Mutation of F 9 Gene in Hemophilia B Patients

Background: Hemophilia B is an X-linked recessive disorder caused by mutations in the coding sequence of F9 gene, leading to dysfunctional Factor IX (FIX) protein. Objectives: This study is to identify novel and recurrent mutations in hemophilia B patients. Method and Materials: In this study, 9 hemophilia B patients were screened on 8 exons using polymerase chain reaction (PCR) and direct sequencing. Results: We identified 6 point mutations, including 4 missense mutations and 2 nonsense mutations. One of the six point mutations is a novel mutation (NM_000133.3:c.230T>G) which has not been reported previously in hemophilia B database. Single nucleotide transversion of Thymine to Guanine occurs at nucleotide position 230, leading to amino acids substitution from Valine to Glycine at codon 77 in Gla domain. This amino acid substitution affects the protein structure and function in the Gla domain of FIX protein. Seven prediction tools were shown highly consistent result in predicting this novel mutation. Conclusion: In this study, all point mutations were found in the coding sequence especially exon 2, exon 5 and exon 8 and distributed among Gla domain, EGF2 domain and SP domains. Novel mutation c.230T>G occurred at exon 2 of F9 gene which has damaging impact to decrease the stability of protein structure and dysfunction in Gla domain of FIX protein.

Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant.

BackgroundPhlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation.MethodsTargeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi.ResultsRecombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC).ConclusionsThe finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.

MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type

Metaphyseal dysplasia, Spahr type (MDST; OMIM 250400) was described in 1961 based on the observation of four children in one family who had rickets-like metaphyseal changes but normal blood chemistry and moderate short stature. Its molecular basis and nosologic status remained unknown. We followed up on those individuals and diagnosed the disorder in an additional member of the family. We used exome sequencing to ascertain the underlying mutation and explored its consequences on three-dimensional models of the affected protein. The MDST phenotype is associated with moderate short stature and knee pain in adults, while extra-skeletal complications are not observed. The sequencing showed that MDST segregated with a c.619T>G single nucleotide transversion in MMP13. The predicted non-conservative amino acid substitution, p.Trp207Gly, disrupts a crucial hydrogen bond in the calcium-binding region of the catalytic domain of the matrix metalloproteinase, MMP13. The MDST phenotype is associated with recessive MMP13 mutations, confirming the importance of this metalloproteinase in the metaphyseal growth plate. Dominant MMP13 mutations have been associated with metaphyseal anadysplasia (OMIM 602111), while a single child homozygous for a MMP13 mutation had been previously diagnosed as "recessive metaphyseal anadysplasia," that we conclude is the same nosologic entity as MDST. Molecular confirmation of MDST allows distinction of it from dominant conditions (e.g., metaphyseal dysplasia, Schmid type; OMIM # 156500) and from more severe multi-system conditions (such as cartilage-hair hypoplasia; OMIM # 250250) and to give precise recurrence risks and prognosis.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant

BackgroundPhlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation.MethodsTargeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi.ResultsRecombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC).ConclusionsThe finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.

Nanopore Quantitation of Cancer BRAF Driver Mutation Facilitated by a DNA Interstrand Merculock

Driver mutations are a special type of genetic alterations that are causally correlated with oncogenesis. Accurate detection of the presence of driver mutations is extremely useful for early cancer diagnosis. BRAF Serine/threonine-protein kinase B-raf) has a predominant driver mutation V600E, which occurs with the highest incidence mainly in melanoma, colorectal and thyroid cancers, and in other cancers. Currently the BRAF pathway has become a drug arget for molecular therapy. Here we devise a novel nanopore single-molecule assay to accurately detect this driver mutation. The BRAF V600E gene involves a single-nucleotide transversion T1799A in the sense strand, and A1799T in the antisense strand. We selected the antisense strand as the target. Upon hybridization with an optimized probe that contains a thymine at the mutation site, the target•probe complex can form a T-T mismatch. The nanopore single-molecule sensor can be used to visually discriminate this T-T mismatch bound with a mercury ion (Hg2+). This is because the Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in A-T base pair between the normal BRAF gene and the same probe, suggesting that the MercuLock acts as a fingerprint of the mutant DNA. By counting the frequency of MercuLock blocking events in the nanopore, we can quantize trace amount of mutant DNA in the mixture. This approach can be adapted to the detection of any thymine-involved driver mutations and single nucleotide polymorphisms (SNPs) for cancer detection.

Dialects of the DNA Uptake Sequence in Neisseriaceae

In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation in bacteria, and define the phylogenetic relationships within the Neisseriaceae family.

Mutation at Codon 198 Of Tub2 Gene for Carbendazim Resistance in Colletotrichum Gloeosporioides Causing Mango Anthracnose in Thailand

Screening of field isolates of Colletotrichum gloeosporioides from various mango cultivars from markets and orchards in Thailand identified 113 carbendazim-resistant isolates. Isolates with a highly-resistant phenotype (HR) grew well on Potato Dex- trose Agar (PDA) amended with carbendazim even at ≥500 mg/l. Isolates with carbendazim-resistant phenotype had a conspicuous mutation at a particular site in the β-tubulin (TUB2) gene sequence. The sequence of TUB2 in HR isolates showed a single nucleotide transversion of adenine to cytosine, resulting in a substitution at codon 198 from glutamic acid (GAG) in wild type to alanine (GCG) in HR isolates. This is the first report of the molecular determination of field isolates for benzimidazole fungicide resistance in Southeast Asia in C. gloeosporioides causing mango anthracnose.

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes infective to humans, and its implications

The high-resolution analysis of genetic variation has major implications for the identification of parasites and micro-organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR-based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single-nucleotide transversion for amplicons of approximately 1 kb in size. The high-throughput potential and relatively low-cost of REF make it a particularly useful tool for large-scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single-copy genes as well as a wide range of other protozoan and metazoan parasites.

Luminescence-based colorimetric discrimination of single-nucleotide transversions by the combined use of the derivatives of DOTA-conjugated naphthyridine and its terbium complex

We newly synthesized a nucleobase-binding ligand, ND-DOTA, in which 2-amino-5,7-dimethyl-1,8-naphthyridine (ND) was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DOTA) via an amide linker, and found that its terbium(III) complex (ND-DOTA-Tb) showed green emission based on an energy transfer from the naphthyridine moiety to Tb 3+. The blue emission of ND-DOTA was selectively quenched by adding abasic site-containing DNA duplexes that have pyrimidine bases opposite to the abasic site. In contrast, at the same excitation wavelength, ND-DOTA-Tb showed green emission independently of the bases opposite to the abasic site. Thus, a mixed solution of ND-DOTA and ND-DOTA-Tb enabled the luminescence-based colorimetric discrimination of single-nucleotide transversions with the naked eye at a single excitation wavelength.

TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping

Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.

Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant beta2-microglobulin gene.

Two siblings, products of a consanguineous marriage, were markedly deficient in both albumin and IgG because of rapid degradation of these proteins, suggesting a lack of the neonatal Fc receptor, FcRn. FcRn is a heterodimeric receptor composed of a nonclassical MHC class I alpha-chain and beta(2)-microglobulin (beta(2)m) that binds two ligands, IgG and albumin, and extends the catabolic half-lives of both. Eight relatives of the siblings were moderately IgG-deficient. From sera archived for 35 years, we sequenced the two siblings' genes for the heterodimeric FcRn. We found that, although the alpha-chain gene sequences of the siblings were normal, the beta(2)m genes contained a single nucleotide transversion that would mutate a conserved alanine to proline at the midpoint of the signal sequence. Concentrations of soluble beta(2)m and HLA in the siblings' sera were <1% of normal. Transfection assays of beta(2)m-deficient cultured cells with beta(2)m cDNA indicated that the mutant beta(2)m supported <20% of normal expression of beta(2)m, MHC class I, and FcRn proteins. We concluded that a beta(2)m gene mutation underlies the hypercatabolism and reduced serum levels of albumin and IgG in the two siblings with familial hypercatabolic hypoproteinemia. This experiment of nature affirms our hypothesis that FcRn binds IgG and albumin, salvages both from a degradative fate, and maintains their physiologic concentrations.

Genetic polymorphism of the caprine kappa casein gene.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction-restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

Tay-Sachs disease: Intron 7 splice junction mutation in two Portuguese patients

A single nucleotide transversion (G → C) in the 5' donor site of intron 7 of the β-hexosaminidase α-chain gene was identified in two Portuguese patients with infantile Tay-Sachs disease. One patient was found to be homozygous and the other a compound heterozygote with the four-base insertion in exon 11 on the other allele. In fibroblasts from the homozygous patient the β-hexosaminidase α mRNA was observed as a nearly undetectable fast migrating band. Through cDNA-PCR amplification and hybridization with full length a cDNA several fragments of smaller size than the normal transcript were detected, most of them lacking exon 7. We propose that this point mutation in the 5′ donor site of intron 7 of the β-hexosaminidase α-chain gene is responsible for an inefficient and abnormal processing of the mutant transcript, resulting in functional abnormality.

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Mice harboring the waved-1 (wa-1) and waved-2 (wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-alpha-deficient mice, and wa-1 and TGF-alpha were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by > 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-alpha mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-alpha-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.