Adenine Mutation Research Articles

Study of HOXB13 Gene Variants in Prostate Cancer Patients.

Prostate cancer (PC) is a prevalent malignancy with a significant hereditary component. The HOXB13 gene, encoding a transcription factor involved in prostate development, has been implicated in PC risk. The objective of this study was to assess the existence of HOXB13 mutations in PC patients. The retrospective study included 33 PC patients and 23 controls. Demographic data, family history, and smoking habits were recorded. Prostate-specific antigen (PSA) levels were measured. We investigated the second exon of HOXB13 after extracting genomic DNA from blood samples for mutations using polymerase chain reaction and Sanger sequencing. PC patients had a higher mean age (64.7 years), more frequent positive family history (63.64%, N = 21), and higher smoking prevalence (60.61%, N = 20) compared to controls. PSA levels were significantly elevated in patients (76.58 ng/ml) versus controls (7.22 ng/ml). HOXB13 mutations, including thymine (3.03%, N = 1), guanine (27.27%, N = 9), and adenine (33.33%, N = 11) mutations, were observed in patients, while no mutations were found in controls. PC patients had higher mean age, more positive family histories, higher smoking rates, and elevated PSA levels. HOXB13 mutations were significantly higher in patients compared to controls. These findings emphasize the roles of HOXB13, age, family history, smoking, and PSA in PC risk stratification.

Extreme hypernatremia after a laparoscopic hysterectomy and bilateral salpingo-oophorectomy: a case report and literature review.

Congenital nephrogenic diabetes insipidus (NDI) primarily arises from an X-linked recessive inheritance caused by mutations in the AVPR2 gene, which is responsible for approximately 90% of cases. This condition has an incidence rate of 4-8 per million male live births, with females being much less frequently affected. Symptoms typically manifest shortly after birth, predominantly in males. The key clinical features of NDI include excessive urination (polyuria), compensatory excessive thirst (polydipsia), cognitive impairment, consistently low urine specific gravity, dehydration, and imbalances in electrolyte levels. This case study highlights an unusual occurrence of NDI in a 50-year-old Chinese woman attributed to a mutation in the AVPR2 gene. For more than a year, she had been suffering from excessive urination and severe thirst. The patient, who had undergone surgery for cervical cancer, developed polyuria and hypernatremia postoperatively. Initial laboratory analyses revealed normal blood sodium and chloride levels but reduced urine osmolality and specific gravity. Imaging assessments revealed no irregularities. To validate the diagnosis of NDI, she participated in a water deprivation and vasopressin test. Subsequent genetic tests revealed a thymine (T) to adenine (A) mutation, leading to a missense mutation in the AVPR2 gene. As part of her treatment, she was placed on a low-sodium diet and prescribed oral hydrochlorothiazide and indomethacin for 1 month, resulting in a marked improvement in her symptoms. To the best of our knowledge, this is the first documented case of NDI diagnosed postoperatively in an older female patient with AVPR2 heterozygosity. This case highlights an unusual instance of an X-linked recessive clinical presentation of NDI in an elderly female patient. This study also underscores the importance of conducting water deprivation, vasopressin tests, and genetic testing in establishing the underlying cause for individuals diagnosed with NDI.

Phenotypic and Genotypic Characterization of Sheep Breeds in Diverse Habitats of Baluchistan Province through the Analysis of the vertnin Gene

This study was conducted to identify the vertnin gene in four different sheep breeds of Balochistan by studying their genetic characteristics and analyzing molecular markers. For this purpose, four different sheep breeds, Balochi, Rakhshani, Harnai, and Bibrik, were selected. A total of 10 animals from each breed were chosen to study the impact of breed and the vertnin gene on carcass features. The selection of animals was based on their phenotypic characteristics such as age and teeth. Blood samples of 10 ml were collected and transported to the Laboratory of Molecular Genetics at the Faculty of Veterinary and Animal Sciences, LUAWMS Uthal, Balochistan. Genomic DNA (gDNA) extraction was carried out using a modified inorganic method. The results showed that Balochi had a change in amino acid cytosine (C) > into thymine (T) mutation, Rakhshani had a change in guanine (G) > into adenine (A) mutation, Bibrik had a change in adenine (A) > into cytosine (C) mutation, and Harnai had a change in thymine (T) > into cytosine (C) mutation. The maximum divergence was 0.15715 bp in relation to the comparison in all studied sheep breeds. The phylogenetic results showed that the Balochi breed has 95–97% similarities with the other breeds when sequences were compared. The results revealed that the gel-electrophoresis bands of the Balochi breed were slightly higher, respectively. It was concluded that the vertnin gene exists in different breeds of sheep and might show a broad range of differences, even with similar DNA sequences. However, the purity based on the vertnin gene is at high risk, as a notable difference was found between the Balochi and Harnai sheep.

Molecular Detection of Glucose-6-Phosphate Dehydrogenase Deficiency in Katsina State, Northern Nigeria

Aims: To determine the prevalence of glucose-6-phosphate dehydrogenase deficiency and its variant (G6PD A-) among children diagnosed with Plasmodium falciparum malaria in Katsina state, Nigeria.
 Study Design: Cross-Sectional Studies.
 Place and Duration of Study: General Hospitals Katsina, Dutsin-ma, Daura, Baure, Malumfashi and Funtua of Katsina state, Nigeria from June, 2020 to December, 2020.
 Methodology: A total of 200 blood samples were collected from the study subjects after getting the ethical approval and informed consent. Their socio-demographic information and clinical presentations were also noted with the aid of questionnaire. G6PD deficiency was detected using G6PD qualitative test. Molecular characterization of African A- Variants was carried out using PCR and Sanger sequencing. Phylogenetic studies were carried out to analyze the relationship between the types of mutations found in Nigeria and other countries.
 Results: The G6PD qualitative test shows that 35(17.5%) samples were G6PD deficient which indicates significant association (P<0.05) between G6PD and malaria. The PCR and sequence analysis of the 35 G6PD deficient samples shows the presence of G202A mutations in only 7(20.0%) samples. However, the BLAST analysis of the nucleotide sequences has shown 98.73% - 100% homology with other sequences of G6PD from the NCBI database. The bioinformatics analysis revealed G6PD mutations which indicate a Guanine to Adenine mutations at amino acid number 68 substitution of valine to methionine.
 Conclusion: This study has shown a high prevalence of G6PD deficiency among children diagnosed with Plasmodium falciparum malaria in Katsina State, North-western Nigeria. Polymerase Chain Reaction, NCBI blast, Phylogenetic and Bioinformatics analysis of the deficient samples shows that G202A mutation in relation to the deficient children was not statistically significant (p>0.05), hence does not appear to have a role in G6PD deficiency among children in the selected area of Katsina state, Nigeria though our findings were limited by the small sample size.

Genome-wide association mapping and examination of possible maternal effect for the pace trait of horses.

Previously, a single nucleotide polymorphism (SNP) related to gait type was identified at position 22999655 of chromosome 23 in the coding region of DMRT3 (DMRT3:Ser301Ter) by showing that a cytosine (C) to adenine (A) mutation of this SNP induced pace in the Icelandic horse. We investigated the effect of DMRT3:Ser301Ter on the gait of Hokkaido Native Horses, a Japanese native breed, and examined genetic factors other than DMRT3 by exploring genome-wide SNPs related to gait determination. All animals exhibiting pace were AA for DMRT3:Ser301Ter, confirming the association of DMRT3:Ser301Ter with gait determination; however, 14.3% of the animals exhibiting trot also had AA for DMRT3:Ser301Ter, suggesting the presence of another factor(s) cooperatively working with DMRT3:Ser301Ter for gait determination. SNPs on chromosomes 13 and 23 were detected by genome-wide association analysis (false discovery rate <0.05), although SNPs on chromosome 23 were all located in the vicinity of DMRT3:Ser301Ter, confirming the association with DMRT3. A genome-wide association study targeting only animals with AA for DMRT3:Ser301Ter to examine genetic factors cooperatively working with DMRT3:Ser301Ter for gait determination suggested associations of 23 SNPs on six chromosomes. In a series of analyses of the effect of a maternal factor (dam's gait) on gait determination, the effect was suggested in comparison of the frequencies of exhibiting pace in gait checks in only two animal groups having dams with different DMRT3:Ser301Ter genotypes (P<0.05), suggesting that the gait of the dam does not have a major effect on whether progeny homozygous for the DMRT3:Ser301Ter mutation will preferentially pace or trot.

Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars.

The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (SSIIaI). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Abstract A26: High frequency of Cytosine to Adenine mutations in neuroblastoma correlates with genomic aberrations in 8-Oxo-Guanine repair pathway

Abstract Background: Somatic mutations can be grouped into 96 tri-nucleotide patterns, termed mutational signatures. These signatures can provide insights into underlying processes that fuel the evolution of tumor cells, and may expose therapeutic intervention avenues. Here we investigated the mutational signature of 84 whole genome sequenced neuroblastoma cases. Results: Mutational signature analysis revealed an extreme bias towards Cytosine to Adenine (CtoA) mutations in a majority of neuroblastoma tumors. This seems to be a unique feature of neuroblastoma. CtoA mutations are primarily found in high stage tumors and are therefore associated with a poor prognosis. Oxidative stress is known to be a source for such mutations, which can accumulate due to defects in the 8-Oxo-Guanine repair pathway genes OGG1, MUTYH and MTH1. We could show a strong correlation between CtoA mutations and chromosomal loss of MTH1, OGG1 and MUTYH. Besides copy number losses, sequencing also revealed a tumor with a homozygous mutation in OGG1 and this tumor showed a very strong bias towards CtoA mutations. Modified alkaline comet assays were performed for quantification of 8-Oxo-Guanine content present in the DNA of neuroblastoma cell lines. These comet assays showed that neuroblastoma cell lines with loss of either OGG1 or MUTYH, had higher 8-Oxo-Guanine content than cell lines without these losses. 8-Oxo-Guanine content is correlated with CtoA mutation frequency in short term cultured neuroblastoma organoids. Overexpression of OGG1 or MUTYH in neuroblastoma cell lines with losses of OGG1 or MUTYH respectively leads to rescue of the phenotype and a decrease in the amount of 8-Oxo-Guanine in the DNA. We are currently performing a compound screen to test the efficacy of 352 compounds functioning in the DNA damage repair in cell lines with and without MUTYH expression, and were testing compounds that further interfere in this specific branch of DNA damage repair including the newly developed MTH1 inhibitors. Conclusion: We identified a subset of neuroblastoma tumors with a high CtoA mutation frequency, which correlates with losses of glycosylases involved in the repair of CtoA mutations. 8-Oxo-Guanine levels are elevated in cell lines with loss of OGG1 or MUTYH, which can be rescued by overexpression of OGG1 or MUTYH respectively. Citation Format: Anne Hakkert, Marli E. Ebus, Rogier Versteeg, Caron N. Huib, Jan Koster, Jan J. Molenaar. High frequency of Cytosine to Adenine mutations in neuroblastoma correlates with genomic aberrations in 8-Oxo-Guanine repair pathway [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A26.

BioSpotlight / Citations

BioTechniquesVol. 60, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114398AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Tagging along with carbonylated proteinsCarbonylation of proteins is a biomarker for oxidative stress that is traditionally studied using two complementary techniques: immunoblotting and tandem mass spectroscopy. For both of these methods, tagging of the carbonylated protein using probes that form a covalent bond with the reactive carbonyl plays an important role. In this month's issue, Timothy J. Griffin and colleagues from the University of Minnesota, along with researchers at Thermo Fisher, introduce a novel multifunctional tagging reagent for analysis of carbonylated proteins. This derivative of the tandem mass tag (TMT) isobaric labeling reagents, called aminoxyTMT, contains a carbonyl-reactive aminooxy group. The authors demonstrate that, in combination with the anti-TMT antibody, aminoxyTMT labeling of carbonylated proteins allows for their visualization in immunoblots and their enrichment from complex mixtures. When used for tandem mass spectroscopy, aminoxyTMT enables the identification of carbonylated proteins and the sites of their modification as well as the relative quantification of the carbonylation state.See “AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation”dPCR samples can't stand the heatDigital PCR (dPCR) has greatly facilitated the accurate quantification of DNA mutations in tissue samples. Fragmentation of intact genomic DNA samples is required prior to dPCR, but there has been no study of the effects of different fragmentation methods on the performance of dPCR point mutation assays. In this issue of BioTechniques, Muneesh Tewari and her group at the University of Michigan examined three methods of fragmentation: heating, sonication, and restriction digestion. Wild-type genomic DNA treated by each method was analyzed by droplet dPCR to determine the wild-type and mutant allele counts for the KRAS and BRAF genes. While heating to 95°C is the easiest fragmentation method, it resulted in a high background of mutation detection for particular KRAS mutations compared to the other two methods. This effect appeared to be due to heat-induced mutations and specifically affected dPCR assays detecting guanine to adenine (G>A) mutations. Fragmentation by heating also overestimated gene copy number. These results indicate that researchers should be careful about which DNA fragmentation method they select for their dPCR-based mutation detection assays.See “Mutant DNA quantification by digital PCR can be confounded by heating during sample preparation”Measuring gene regulationRelating transcription factor binding to specific patterns of gene expression has proven to be difficult, particularly at the single-cell level. But now, Sepulveda et al. describe a workflow for measuring gene regulation in individual cells. Using the lysogeny maintenance promoter of phage lambda as a model system, the authors first simultaneously measured concentrations of the lambda repressor CI in single cells, as well as numbers of mRNA transcripts from the promoter. To measure CI concentrations, they used an immunofluorescence assay, while single-molecule fluorescence in situ hybridization (FISH) provided measurements of promoter activity. By then modeling the CI concentration and transcript data, the researchers were able to define activity patterns that corresponded to specific CI binding configurations and uncover unique insights into promoter state switching. While applied initially to the lysogeny maintenance promoter, this approach should be adaptable to other genetic systems for promoter binding and gene expression studies.Sepulveda et al. 2016. Measurement of gene regulation in individual cells reveals rapid switching between promoter states. Science. 351:1218-1222.Tissue bioprinting gets thickThree-dimensional bioprinting advances have made it possible to create complex tissue architectures in the lab. But more development is needed to fully realize the potential of this technique and use it to faithfully mimic the structure and longevity of human tissue. Now, Kolesky et al. report a new technique for 3-D bioprinting that allows long-term perfusion of thicker tissues on chips. To generate vascularized tissues within perfusion chips, they first printed silicone ink on the glass surface. After this, a cell-laden ink along with ink composed of a triblock copolymer was printed on the chip. The result was a network of interconnected channels lined with cells, which could be perfused over an extended period using external pumps. The authors validated their printing method by creating a simple tissue construct composed of two parallel channels embedded in a fibroblast matrix and then a more complex, heterogenous tissue microenvironment >1 cm thick. This new bioprinting approach is another step towards creating more complex tissue architectures in the lab that can be used for ex vivo and, eventually, in vivo applications.Kolesky, D. et al. 2016. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl. Acad. Sci. 113: 3179-3184.FiguresReferencesRelatedDetails Vol. 60, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2016 Information© 2016 Author(s)PDF download

Mutant DNA Quantification by Digital PCR Can Be Confounded by Heating During DNA Fragmentation

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

Tracking the genomic evolution of esophageal adenocarcinoma through neoadjuvant chemotherapy.

Esophageal adenocarcinomas are associated with a dismal prognosis. Deciphering the evolutionary history of this disease may shed light on therapeutically tractable targets and reveal dynamic mutational processes during the disease course and following neoadjuvant chemotherapy (NAC). We exome sequenced 40 tumor regions from 8 patients with operable esophageal adenocarcinomas, before and after platinum-containing NAC. This revealed the evolutionary genomic landscape of esophageal adenocarcinomas with the presence of heterogeneous driver mutations, parallel evolution, early genome-doubling events, and an association between high intratumor heterogeneity and poor response to NAC. Multiregion sequencing demonstrated a significant reduction in thymine to guanine mutations within a CpTpT context when comparing early and late mutational processes and the presence of a platinum signature with enrichment of cytosine to adenine mutations within a CpC context following NAC. Esophageal adenocarcinomas are characterized by early chromosomal instability leading to amplifications containing targetable oncogenes persisting through chemotherapy, providing a rationale for future therapeutic approaches. This work illustrates dynamic mutational processes occurring during esophageal adenocarcinoma evolution and following selective pressures of platinum exposure, emphasizing the iatrogenic impact of therapy on cancer evolution. Identification of amplifications encoding targetable oncogenes maintained through NAC suggests the presence of stable vulnerabilities, unimpeded by cytotoxics, suitable for therapeutic intervention.

Homozygous variant of UGT1A1 gene mutation and severe neonatal hyperbilirubinemia

The aim of the present study was to compare, in a case-control study, the prevalence of nucleotide 211 guanine to adenine (G-->A) mutation of uridine diphosphoglucuronosyl transferase (UGT1A1) gene in Malaysian Chinese newborns with and without severe hyperbilirubinemia (total serum bilirubin >250 micromol/L during first 48 h of life or > or =300 micromol/L thereafter), and to determine whether this mutation was a significant risk factor associated with severe hyperbilirubinemia. Seventy-four term infants of Chinese descent admitted with severe hyperbilirubinemia were recruited. Infants without severe hyperbilirubinemia (n = 125) were randomly selected from among healthy Chinese term infants. UGT1A1 nucleotide 211 polymorphism was assayed using the Taqman single nucleotide polymorphism genotyping method. Using gestational age, types of feeds, G6PD mutation, G6PD enzyme levels, and UGT1A1 gene mutation status as independent variables, logistic regression analysis was carried out to determine the significant risk factors associated with severe hyperbilirubinemia. UGT1A1 gene mutation was significantly more common among hyperbilirubinemic infants (39.2%) than controls (25.6%; P = 0.04). Gestational age (adjusted odds ratio [OR], 0.7; 95% confidence intervals [CI]: 0.5-0.9; P = 0.01), G6PD mutation (adjusted OR, 7.2; 95%CI: 2.7-19.0; P < 0.0001), exclusive breast-feeding (adjusted OR, 11.7; 95%CI: 2.7-49.9; P = 0.001), and homozygous variant of UGT1A1 gene mutation (adjusted OR, 32.2; 95%CI: 3.8-273.2; P = 0.001) were significant risk factors. Heterozygous variant of UGT1A1 gene mutation, actual levels of G6PD enzyme, and mixed feeding were not. Homozygous variant of nucleotide 211 G-->A mutation of UGT1A1 gene is a significant risk factor associated with severe hyperbilirubinemia among Malaysian Chinese newborns.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4–5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman ® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

The Signal Sequence Coding Region Promotes Nuclear Export of mRNA

In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.

Estimating the Relative Contribution of dNTP Pool Imbalance and APOBEC3G/3F Editing to HIV EvolutionIn Vivo

The human immunodeficiency virus (HIV) has a genome that is rich in adenine, and its rapid evolution shows an observed bias of guanine (G) to adenine (A) mutations. Two mechanisms have been proposed to explain these properties: (1) an imbalance in dNTP pool concentrations which drives the misincorporation process during reverse transcription, and (2) cytidine deamination by the APOBEC3G/3F restriction factor, causing G to A mutations most notably in specific dinucleotide contexts. Although crucial to understanding HIV evolution, current estimates on misincorporation bias during the replication cycle are based on scarce in vitro measurements. In this work, HIV partial pol sequences obtained for drug resistance testing purposes are analyzed using likelihood methods to estimate various models of HIV misincorporation bias in vivo. The technique is robust to selection on the amino acid sequence and selection against CpG dinucleotides. A model where misincorporations are explained only by an imbalance in dNTP pool concentrations, together with a preference for transitions versus transversions, explained 98% (95% confidence interval [C.I.] 93-100) of the observed variation in freely estimated misincorporation rates. Although dinucleotide context was responsible for variation in misincorporation probabilities, this variation was not specific for G to A mutations implying that the footprint of APOBEC3G/3F editing could not be detected. These results indicate that an imbalance in dNTP pool concentrations explains most of the bias in HIV nucleotide misincorporations, while the effect of editing by APOBEC3G/3F on HIV evolution, based on its dinucleotide specificity, could not be observed in this study.

Thrombophilia and Preeclampsia: The Evidence So Far

The Role of Thrombophilias Pregnancy is a hypercoagulable state. The field of thrombophilia, the tendency to thrombosis, has been developed rapidly and has been linked to many aspects of pregnancy. Recently, it has been determined that severe pregnancy complications such as severe preeclampsia intrauterine growth retardation (IUGR), abruptio placentae, and intrauterine fetal death (IUFD) are associated with thrombophilia. Thrombophilias are inherited or acquired conditions that predispose an individual to thromboembolism. Deficiencies of protein S, C, and antithrombin are rare and each of them is found in approximately 3% of patients with thrombosis. Three important inherited thrombophilias were discovered, which are responsible of the majority of thromboembolic events in patients with no otherwise apparent risk for thrombosis. Resistance to activated protein C caused by an adenine 506 guanine (A506G) mutation in factor V (factor V Leiden) has been linked with an increased risk for venous thromboembolism. Heterozygosity for the factor V (FV) Leiden mutation is found in approximately 5% of the population and the mutation is responsible for 20% to 30% of venous thromboembolism events. A recently described guanine 20210 adenine mutation in prothrombin is associated with higher plasma prothrombin concentrations and increased risk for venous thromboembolism and cerebral vein thrombosis. Homozygosity for the cytosine 677 thymine (C677T) mutation in methylenetetrahydrofolate reductase (MTHFR) results in decreased synthesis of 5-methyltetrahydrofolate, the primary methyl donor in the conversion of homocysteine to methionine, and the resulting increase in plasma homocysteine concentrations is a risk factor for thrombosis. The mutation is responsible for reduced MTHFR activity and is the most frequent cause of mild hyperhomocystinemia and can be found in 5% to 15% of the population. Homocysteine is an independent risk Correspondence: Michael J. Kupferminc, MD, Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. E-mail: tmcobgyn@tasmc.health. gov.il

High prevalence of the prothrombin gene mutation in women with intrauterine growth retardation, abruptio placentae and second trimester loss

It has been reported recently that obstetric complications are associated with thrombophilias. Our objective was to investigate the association between pregnancy complications and the guanine 20210 adenine (G20210A) mutation in prothrombin gene. Two hundred and twenty-two women (study group) with obstetric complications were tested for the prothrombin mutation. Indications for testing were: severe preeclampsia, mild preeclampsia, intrauterine growth retardation, severe abruptio placentae, unexplained stillbirth, second trimester loss, and three or more consecutive spontaneous abortions. We also tested 156 healthy women who had at least one normal pregnancy and comprised the control group. Demographic data of the study and control groups were similar. Twenty-eight women of the study group (13%) were found to be heterozygous carriers of the 20210 variant of the prothrombin gene compared to five (3.2%) of the control group, p=0.001, odds ratio (OR) 2.9; 95% confidence interval (CI) 1.3-6.5. Compared to the control women, the prothrombin gene mutation was significantly more prevalent in women with IUGR, abruptio placentae, and second trimester loss but not in women with mild or severe preeclampsia, stillbirth and habitual abortion. Our data demonstrate that the mutation in the prothrombin gene is associated with specific pregnancy complications.

Lattice corneal dystrophy. Detection of a point mutation in the kerato-epithelin gene

Lattice dystrophy is an autosomal-dominantly inherited disease. A mutation of the gene coding for kerato-epithelin has been found in patients with this stromal dystrophy. In codon 124 a Guanine to Adenine mutation of the nucleotide 417 has been described. We looked for this mutation in a family with lattice dystrophy treated in our clinic. Using primers specific for kerato-epithelin gene, we amplified the cDNA extracted from lymphocytes of two patients suffering from lattice dystrophy. The polymerase chain reaction (PCR) products were subcloned and sequenced. Guanine to Adenine mutations, as published were detected in both of our patients at codon 124. We found the published mutation in both of our patients, indicating that this Guanine to Adenine exchange is pathognomonic for lattice dystrophy.

A trinucleotide deletion together with a base duplication event at codon 439 in the human tyrosinase gene identifies a mutational hotspot

Molecular analysis of the human tyrosinage gene in two patients suffering from a temperature-sensitive form of albinism has identified a thymine triplet deletion at codon 439 which is accompanied by a duplication of the immediately preceding cytosine residue. This results in a two base pair frame shift leading to premature termination at codon 448, giving a truncated protein. Its relationship to other mutations in tyrosinase and the possible cause are discussed. The temperature-sensitive phenotype is due to the guanine to adenine mutation at codon 422, known to generate a temperature-sensitive enzyme. The CTTT at F 439 in tyrosinase is also present at F 508 in CFTR, the main mutation causing cystic fibrosis.

Predominance of the mutation at 1138 of the cDNA for the fibroblast growth factor receptor 3 in Japanese patients with achondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) has recently been identified as a putative gene for achondroplasia. Since a guanine to adenine mutation at 1138 of the cDNA for FGFR3 had been identified in most of the patients in Western population, we examined 13 Japanese patients to see if they also share the same mutation. Specific endonuclease digestion of the amplified coding sequence for the transmembrane domain of the FGFR3 revealed that the 12 patients have the G to A change at 1138, while the other had the G to C substitution at the same point, both of which result in G380A substitution. As far as we studied, the homogeneity of the point mutation at 1138 is also authentic to Japanese patient as well as Western patients.

Cholesteryl ester transfer protein deficiency: Identification in the Chinese

Cholesteryl ester transfer protein (CETP) regulates cholesterol content in high-density lipoproteins (HDL) through the exchange of cholesteryl ester in HDL and triglyceride in triglyceride-rich lipoproteins. Three types of CETP deficiency have so far been reported, all in Japanese individuals. Among them, two types of mutation in the CETP gene are relatively common: (1) a guanine to adenine mutation in the intron 14 splicing donor site (114A), and (2) an Asp 442 to Gly (D442G) mutation. The 114A mutation has an estimated allele frequency of 0.81% in the Japanese. It elevates the plasma HDL-cholesterol level strikingly: typically, >120 mg/dl for homozygotes and 80-110 mg/dl for heterozygotes. In contrast, the D442G mutation has a higher estimated allele frequency (4.62%), but has a mild effect on the HDL-cholesterol level: typically, >80 mg/dl for homozygotes and 50-100 mg/dl for heterozygotes. We screened Chinese individuals for the two CETP gene mutations. 7 refs., 1 tab.