Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position.     

Essential Genes and Deficiencies in the UNC-22 IV Region of CAENORHABDITIS ELEGANS

Five formaldehyde-induced deficiencies that uncover unc-22 IV, a gene affecting muscle structure in the nematode Caenorhabditis elegans were isolated and positioned. The largest deficiency, sDf2, extends in both directions from unc-22 and is approximately 1.0–2.0 map units in length. The other four deficiencies, sDf7, sDf8, sDf9 and sDf10, are all smaller than sDf2 and are located within the region uncovered by this deficiency. Thirty-seven ethyl methanesulfonate-induced lethal and sterile mutations linked to unc-22 were isolated and tested for complementation with sDf2. Nineteen lethal mutations failed to complement sDf2. Sixteen of these were further positioned by recombination mapping and also by deficiency mapping with sDf7, sDf8, sDf9 and sDf10. These sixteen mutations define 11 new essential genes in this region. Eight of the genes lie in a 0.9-map unit interval to the left of unc-22, whereas the three remaining genes lie in a region of about 0.2 map units to the right of unc-22. We believe that two of the essential genes identified in this study, let-56 and let-52, are the adjacent genes on either side of unc-22. The lethal mutations exhibit a wide range of terminal phenotypes: from first stage larva to sterile adult.     

Homoeosis in Drosophila: a new enhancer of polycomb and related homoeotic mutations

A new recessive lethal mutation in Drosophila melanogaster , Enhancer of Polycomb [E(Pc)], and chromosomal deficiencies lacking this locus act as dominant enhancers of the Polycomb mutant syndrome in adults. Thus, although E(Pc)/+ flies are phenotypically normal, this locus is haplo-abnormal with respect to its effect on the Polycomb phenotype. Recombinational and deficiency mapping localize the E(Pc) locus on chromosome 2 proximally and very closely linked (~0.1 map unit) to the engrailed gene. E(Pc) enhances the expression of all Polycomb point mutations examined including that of a deficiency, indicating that this interaction does not depend on the presence of an altered Polycomb gene product. In several respects the mutations extra sex comb, lethal(4)29, and Polycomblike resemble those at the Polycomb locus. In the presence of E(Pc), recessive alleles of extra sex comb and lethal(4)29 are rendered slightly pseudodominant, and the homoeotic effects of Polycomblike heterozygotes are also enhanced. However, E(Pc) does not affect the expression of dominant mutations within the Bithorax gene complex (Cbx) or Antennapedia gene complex (Antp^Ns, Antp^73b, Antp^scx , Antp^EfW15, Scr^Msc) which give homoeotic transformations resembling those of the Polycomb syndrome. Available evidence from the study of adult phenotypes suggests that mutations at E(Pc) do not result in homoeotic changes directly but instead modify the expression of a specific set of functionally related homoeotic variants.     

The involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae

The structure of O-linked acidic oligosaccharide from Saccharomyces cerevisiae was analyzed. The chitinase, exclusively O-glycosylated extracelluar protein, was purified from strains mnn1, mnn1 mnn4, mnn1 mnn6 and Δkre2 and the oligosaccharides were hydrolyzed by O-linked sugar chain specific hydrazinolysis. The mannosylphosphorylated mannotriose (M3-P-M) was detected in strain mnn1, but not in the other three strains (mnn1 mnn4, mnn1 mnn6 and Δkre2). α-Mannosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of mannosylphosphorylated mannotriose revealed that mannosylphosphate was attached to a middle mannose of α-1,2-linked mannotriose. This result indicates that the mnn4 and mnn6 mutations affect the mannosylphosphorylation of O-linked oligosaccharide, together with that of N-linked oligosaccharide. The amount of mannosylphosphorylated mannotriose was 7% of total O-linked oligosaccharides (20% of neutral mannotriose) of chitinase in strain mnn1.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

Hydroxylamine-induced purple adenine (ad-3) mutants in Neurospora crassa. I. Characterization of mutants by genetic tests

The genetic effects of hydroxylamine (HA) on Neurospora crassa were studied in an effort to understand the difference between the results obtained on very simple prokaryotic systems and those obtained with mammalian systems. A 2-component heterokaryon was used to study the inactivation of conidia and the induction of recessive lethal mutations at specific loci and over the entire genome. The heterokaryon is heterozygous for 2 closely linked loci, ad-₃A and ad-₃B, in the ad-₃ region. Specific locus mutations can result from either point mutation or chromosome deletion. The results were as follows: (1) Both homokaryotic and heterokaryotic conidia had multi-hit survival curves, and there was no difference between the survival levels of the two as a function of treatment time. (2) The frequency of recessive lethal mutations in the ad-₃ region increased as the square of treatment time.     

Requirement for Abasic Endonuclease Gene Homologues in Arabidopsis Seed Development

Arabidopsis thaliana has three genes, Ape1L, Ape2, and Arp, that show homology to abasic (apurinic/apyrimidinic) endonuclease genes of bacterial, yeast, or animal cells. In bacteria, yeast, and animals, abasic endonucleases function in base excision repair of oxidized and other modified DNA bases. Here we report that plants with knock-out mutations in any one of Ape1L, Ape2, or Arp show no apparent differences from wild type in growth rate, growth habit, and fertility. However, coincident knock-out mutations in Ape1L and Ape2 are lethal and lead to abortion of developing embryos. Mutations of Arp are not deleterious, even in combination with one of the other two mutations. The results are consistent with the interpretation that the process of base excision repair, involving at least one intact copy of Ape1L or Ape2, is required in the process of embryogenesis.     

DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

Electronic excited molecular oxygen (singlet oxygen, ¹O₂) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by ¹O₂ in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a ¹O₂-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with ¹O₂-induced DNA damage are linked to the context of the damaged sequence.     

Structure-function analysis of the Yhc1 subunit of yeast U1 snRNP and genetic interactions of Yhc1 with Mud2, Nam8, Mud1, Tgs1, U1 snRNA,SmD3 and Prp28

Yhc1 and U1C are homologous essential subunits of the yeast and human U1 snRNP, respectively, that are implicated in the establishment and stability of the complex of U1 bound to the pre-mRNA 5′ splice site (5′SS). Here, we conducted a mutational analysis of Yhc1, guided by the U1C NMR structure and low-resolution crystal structure of human U1 snRNP. The N-terminal 170-amino acid segment of the 231-amino acid Yhc1 polypeptide sufficed for vegetative growth. Although changing the zinc-binding residue Cys6 to alanine was lethal, alanines at zinc-binding residues Cys9, His24 and His30 were not. Benign alanine substitutions at conserved surface residues elicited mutational synergies with other splicing components. YHC1-R21A was synthetically lethal in the absence of Mud2 and synthetically sick in the absence of Nam8, Mud1 and Tgs1 or in the presence of variant U1 snRNAs. YHC1 alleles K28A, Y12A, T14A, K22A and H15A displayed a progressively narrower range of synergies. R21A and K28A bypassed the essentiality of DEAD-box protein Prp28, suggesting that they affected U1•5′SS complex stability. Yhc1 Arg21 fortifies the U1•5′SS complex via contacts with SmD3 residues Glu37/Asp38, mutations of which synergized with mud2Δ and bypassed prp28Δ. YHC1-(1-170) was synthetically lethal with mutations of all components interrogated, with the exception of Nam8.     

Crossover Suppressors and Balanced Recessive Lethals in CAENORHABDITIS ELEGANS

Two dominant suppressors of crossing over have been identified following X-ray treatment of the small nematode C. elegans. They suppress crossing over in linkage group II (LGII) about 100-fold and 50-fold and are both tightly linked to LGII markers. One, called C1, segregates independently of all other linkage groups and is homozygous fertile. The other is a translocation involving LGII and X. The translocation also suppresses crossing over along the right half of X and is homozygous lethal. C1 has been used as a balancer of LGII recessive lethal and sterile mutations induced by EMS. The frequencies of occurrence of lethals and steriles were approximately equal. Fourteen mutations were assigned to complementation groups and mapped. They tended to map in the same region where LGII visibles are clustered.     

A Cytogenetic Analysis of the Chromosomal Region Surrounding the α-Glycerophosphate Dehydrogenase Locus of DROSOPHILA MELANOGASTER

The chromosomal region surrounding the structural gene for α-glycerophosphate dehydrogenase (αGpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. αGpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the αGpdh locus.     

lac Repressor-operator interaction. IX. The binding of lac repressor to operators containing Oc mutations

Representative members of the six classes of operator constitutive (O^c) point mutations, which have been mapped and well characterized in vivo, were crossed into λφ80 lac phages. The phage DNAs containing the O^c mutations were used to measure the affinity of the lac repressor (R) for each O^c operator by determining the half-lives of the different RO^c complexes in vitro. The results provide evidence that: (a) the higher the constitutive level of β-galactosidase in vivo, as the result of an O^c mutation, the lower the affinity of the lac repressor for that O^c operator, with a maximum difference of two orders of magnitude in affinity of the repressor for the highest O^c tested as compared to the wild type O⁺ operator; (b) the six classes of O^c operators appear to be twofold degenerate, in that two members of each class, which were previously distinguished by mapping, have the same affinity for the lac repressor; (c) an inducer and an anti-inducer have the same effect on the RO^c complexes as on the RO⁺ complexes; (d) the relationship between induction ratios in vivo and the binding constant of the repressor for each O^c mutation in vitro does not follow the mass action equation but rather a more complex dependency, which is discussed.These results suggest a functional symmetry in the lac operator.     

Genomic Analyses Reveal Mutational Signatures and Frequently Altered Genes in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.     

gammaTub23C interacts genetically with brahma chromatin-remodeling complexes in Drosophila melanogaster

The brahma gene encodes the catalytic subunit of the Drosophila melanogaster BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, we isolated the dominant-negative Pearl-2 allele of γTub23C. γTub23C encodes one of the two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. γ-Tubulin is a subunit of microtubule organizer complexes. We show that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by γTub23C mutations. The genetic interactions between γTub23C and chromatin-remodeling mutations suggest that γ-tubulin might have a role in regulating gene expression.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Dissolution of Xylose Metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl⁻. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl⁺ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Genome-Wide Association Studies Identify Two Novel BMP15 Mutations Responsible for an Atypical Hyperprolificacy Phenotype in Sheep

Some sheep breeds are naturally prolific, and they are very informative for the studies of reproductive genetics and physiology. Major genes increasing litter size (LS) and ovulation rate (OR) were suspected in the French Grivette and the Polish Olkuska sheep populations, respectively. To identify genetic variants responsible for the highly prolific phenotype in these two breeds, genome-wide association studies (GWAS) followed by complementary genetic and functional analyses were performed. Highly prolific ewes (cases) and normal prolific ewes (controls) from each breed were genotyped using the Illumina OvineSNP50 Genotyping Beadchip. In both populations, an X chromosome region, close to the BMP15 gene, harbored clusters of markers with suggestive evidence of association at significance levels between 1E⁻⁰⁵ and 1E⁻⁰⁷. The BMP15 candidate gene was then sequenced, and two novel non-conservative mutations called FecX^Gr and FecX^O were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (p_FecX^Gr = 5.98E⁻⁰⁶ and p_FecX^O = 2.55E⁻⁰⁸). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecX^Gr/FecX^Gr, LS = 2.50±0.65 versus FecX⁺/FecX^Gr, LS = 1.93±0.42, p<1E⁻⁰³ and FecX^O/FecX^O, OR = 3.28±0.85 versus FecX⁺/FecX^O, OR = 2.02±0.47, p<1E⁻⁰³). Both mutations are located in very well conserved motifs of the protein and altered the BMP15 signaling activity in vitro using a BMP-responsive luciferase test in COV434 granulosa cells. Thus, we have identified two novel mutations in the BMP15 gene associated with increased LS and OR. Notably, homozygous FecX^Gr/FecX^Gr Grivette and homozygous FecX^O/FecX^O Olkuska ewes are hyperprolific in striking contrast with the sterility exhibited by all other known homozygous BMP15 mutations. Our results bring new insights into the key role played by the BMP15 protein in ovarian function and could contribute to a better understanding of the pathogenesis of women′s fertility disorders.     

Direct Evidence on the Contribution of a Missense Mutation in GDF9 to Variation in Ovulation Rate of Finnsheep

The Finnish Landrace (Finnsheep) is a well known high-prolificacy sheep breed and has been used in many countries as a source of genetic material to increase fecundity of local breeds. Analyses to date have indicated that mutations with a large effect on ovulation rate are not responsible for the exceptional prolificacy of Finnsheep. The objectives of this study were to ascertain if: 1) any of 12 known mutations with large effects on ovulation rate in sheep, or 2) any other DNA sequence variants within the candidate genes GDF9 and BMP15 are implicated in the high prolificacy of the Finnish Landrace breed; using material from lines developed by divergent selection on ovulation rate. Genotyping results showed that none of 12 known mutations (FecB^B, FecX^B, FecX^G, FecX^GR, FecX^H, FecX^I, FecX^L, FecX^O, FecX^R, FecG^E, FecG^H, or FecG^T) were present in a sample of 108 Finnsheep and, thus, do not contribute to the exceptional prolificacy of the breed. However, DNA sequence analysis of GDF9 identified a previously known mutation, V371M, whose frequency differed significantly (P<0.001) between High and Low ovulation rate lines. While analysis of ovulation rate data for Finnsheep failed to establish a significant association between this trait and V371M, analysis of data on Belclare sheep revealed a significant association between V371M and ovulation rate (P<0.01). Ewes that were heterozygous for V371M exhibited increased ovulation rate (+0.17, s.e. 0.080; P<0.05) compared to wild type and the effect was non-additive (ovulation rate of heterozygotes was significantly lower (P<0.01) than the mean of the homozygotes). This finding brings to 13 the number of mutations that have large effects on ovulation rate in sheep and to 5, including FecB^B, FecG^E, FecX^O and FecX^GR, the number of mutations within the TGFβ superfamily with a positive effect on prolificacy in the homozygous state.