Base-pair Transitions Research Articles

Force-Field-Dependent DNA Breathing Dynamics: A Case Study of Hoogsteen Base Pairing in A6-DNA.

The Hoogsteen (HG) base pairing conformation, commonly observed in damaged and mutated DNA helices, facilitates DNA repair and DNA recognition. The free energy difference between HG and Watson-Crick (WC) base pairs has been computed in previous studies. However, the mechanism of the conformational transition is not well understood. A detailed understanding of the process of WC to HG base pair transition can provide a deeper understanding of DNA repair and recognition. In an earlier study, we explored the free energy landscape for this process using extensive computer simulation with the CHARMM36 force field. In this work, we study the impact of force field models in describing the WC to HG base pairing transition using meta-eABF enhanced sampling, quasi-harmonic entropy calculation, and nonbonded energy analysis. The secondary structures of both base pairing forms and the topology of the free energy landscapes were consistent over different force field models, although the relative free energy, entropy, and the interaction energies tend to vary. The relative stability of the WC and HG conformations is dictated by a delicate balance between the enthalpic stabilization and the reduced entropy of the structurally rigid HG structure. These findings highlight the impact that subtleties in force field models can have on accurately modeling DNA base pair dynamics and should stimulate further computational investigations into other dynamically important motions in DNA.

An Arg124Cys mutation in transforming growth factor β-induced gene associated with lattice corneal dystrophy type I in a Chinese pedigree.

Purpose:To identify a clinical and genetic form of a large Chinese family with an autosomal-dominant lattice corneal dystrophy type I (LCD I).Methods:The patients’ eyes were examined on the basis of slit-lamp microscopy, and other clinical records were also collected. Genomic DNA was extracted from peripheral leukocytes of the affected patients and their unaffected family members. Each previous reported mutation of the transforming growth factor β-induced gene (TGFBI) gene was amplified by touch-down polymerase chain reaction and directly sequenced to verify the disease-causing mutation.Results:Typical clinical features of LCD I were found by slit-lamp photography in these affected Chinese pedigrees. A heterozygous single base-pair transition from C to T (c.418 C > T), leading to amino acid substitution Arg124Cys (R124C) in the encoded TGFBI protein, was detected in all of the eighteen affected patients. The same mutation was not found in unaffected family members.Conclusion:The R124C mutation hot spot, which was relatively rare in China, was responsible for LCD I in the large family. Molecular genetic analysis of TGFBI gene can offer an accurate diagnosis of patients with lattice corneal dystrophies in the clinical treatment.

Molecular Dynamical Investigation of the 7,8-dihydro-8-oxoguanine Mutation in dsDNA

Background: The oxidization of a Guanine (G) base pair to 7,8-dihydro-8-oxoguanine (OG) is one of the most common DNA mutations. OG mutations can undergo a regular Watson-Crick base-pairing, or a reverse Hoogsteen (HG) base-pairing, especially in OG:A mismatches. While the causes of these mutations are well-understood, the kinetic and energetic characteristics of this new pseudo-base have never been fully investigated, especially at temperatures around biological function (17-37°C). Methods: We created a simulation to derive the Free Energy Surface (FES) of OG:C and OG:A Hoogsteen to Watson-Crick base-pair (bp) transitions under multiple temperatures, relative to 2 collective geometric variables: the dihedral Chi and the pseudo-dihedral CPD angle. To make the simulation, we used the relatively recent Metadynamics algorithms in conjunction with GROMACS 2020.2. Results: The lowest free energy increased linearly with increasing temperatures (17-37°C). Major Chi and CPD rotations at these minima varied heavily for 27°C and 32°C (the largest was seen in the former), but stayed relatively similar for other temperatures, indicating a highly sensitive relationship to temperature, likely due to DNA flexibility, quantum mechanical (QM) effects, and hydrogen bonding. Free energies had a weak negative linear relationship, and free energy hypersurfaces were given for studied temperatures of 17-37°C. Human body temperature (37°C) results were also included and explained. The simulations showed why OG:A Hoogsteen bps often occur in organisms and are energetically preferable to standard Watson-Crick. OG:C HG base pairings are determined to likely be not as common as OG:A HG. Limitations: Future investigations must focus on discovering rate constants of these base-pairs, as time constraints did not permit them to be done here, as well as more QM-focused simulations.

Free-Energy Landscape of a pH-Modulated G·C Base Pair Transition from Watson-Crick to Hoogsteen State in Duplex DNA.

In double-helical DNAs, the most stable Watson-Crick (WC) base pair (bp) can be in thermal equilibrium with much less abundant Hoogsteen (HG) bp by the spontaneous rotation of the glycosidic angle in purine bases. Previous experimental studies showed that in the case of a G·C bp, the population of the transient HG is enhanced as a protonated form (HG+) through the protonation of the cytosine base under weakly acidic conditions. Hence, pH is a key factor that can modulate this transition event from the WC to HG+ bp. In this study, to computationally probe the overall free-energy landscapes of this pH-modulated G·C HG breathing, a comprehensive classical molecular dynamics (MD) simulation protocol is proposed using an enhanced sampling MD in conjunction with the standard thermodynamic integration method. From this MD protocol proposed, the free-energy surfaces of the G·C bp transition from the WC to HG bp were constructed successfully at any pH range, producing pH-dependent free-energy quantities in close agreement with previously reported experimental results. The simulation protocol is expected to provide valuable atomistic insight into the DNA bp transition events coupled with protonation or tautomeric shift in a target bp.

Comparative analysis of genome editing systems, Cas9 and BE3, in silkworms

The clustered regularly interspaced short palindromic repeats (CRISPR) system and the Cas9-derived proteins have been applied to genome editing in various organisms. Traditional Cas9 is typically used to knockout genes or specific DNA fragments based on the generation of double-stranded breaks, whereas nCas9 and dCas9 are fused with effectors to perform base pair transitions or epigenetic modification and regulation. However, this system has off-target effects and can cause genomic structure variations. Here, we comparatively analyzed Cas9 and BE3, an initial base editor based on the nCas9 fusion protein, in silkworms. Our results showed that base editing was superior to Cas9 in silkworm cultured cells. BE3 introduced accurate termination codons, whereas Cas9 did not. Moreover, Cas9 induced chromosome translocation, chromosome fragment repetition, and chromosome fragment deletion, with the deletion frequency reaching up to 4.29%. BE3 was not able to induce these changes in our study. Furthermore, Cas9-derived proteins blocked ribosome advance and mRNA transcription for 9 days, with a 9.40% repression effect by combining with double-stranded DNA when single guide RNAs were targeted in the coding region in silkworms. Overall, our findings established a strategy for choosing suitable editing tools for various applications in different organisms.

Hereditary renal amyloidosis with a variant lysozyme p.Trp82Arg in a Chinese family: case report and literature review

BackgroundLysozyme amyloidosis is a rare hereditary systemic amyloidosis with amyloid deposits in various tissues leading to progressive organ failure. It has been mainly reported in developed countries since 1993. Here we report a lysozyme amyloidosis family with variant lysozyme p.Trp82Arg in a Chinese family.Case presentationThe main clinical manifestation of this case was dominant kidney involvement presenting with proteinuria and decreased renal function. Biopsy of the kidney showed massive amyloid deposits in the glomerular mesangium and subendothelium. Immunohistochemistry and mass spectrometry of renal tissue confirmed the lysozyme nature of the amyloid. DNA sequencing of the peripheral blood leukocytes revealed a single base-pair transition from T to C (TGG/ CGG) of codon 82, leading to the replacement of tryptophan by arginine in the mature protein (p.Trp82Arg). The affected patients in this family also presented with dominant kidney involvement, one of them has been confirmed by IHC and mass spectrometry on his renal biopsy and gene testing as well. As there is no radical therapy for lysozyme amyloidosis, patients were given symptomatic treatment such as antihypertensive drugs and antibiotics. To our knowledge, this is the first report of lysozyme amyloidosis in a Chinese family.ConclusionsHereditary amyloidosis with a variant lysozyme of p.Trp82Arg presented with dominant kidney involvement was firstly reported in a Chinese family.

Abstract 2006: Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways.

Abstract DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR) deficient tumors and convey them with different prognostic and therapeutic outcome upon chemotherapy. Using whole-genome and -exome sequencing, we here characterize the somatic mutation patterns underlying MMR-deficient tumors. In particular, we show that mutation frequencies are >30-fold increased relative to MMR-proficient tumors: insertion-deletion (indel) mutations are largely confined to homopolymers, whereas substitutions mainly consist of single basepair transitions located near indels. Since exonic indels result in loss-of-function frameshift mutations, indels were less frequent in the exome due to negative selection. However, a small fraction of exonic indels was positively selected and occurred as hotspot mutations, preferentially affecting the ‘DNA double-strand break (DSB) repair by homologous recombination (HR)’ pathway (on average, each MMR-deficient tumor contained 2.6 indels in this pathway). We quantified DSB repair by immunofluorescent staining of RAD51 foci in 8 MMR-deficient and 4 MMR-proficient tumor cultures in the presence of the PARP inhibitor olaparib. We did not observe a difference in RAD51 foci formation between MMR-deficient and MMR-proficient tumor cultures in the absence of olaparib (10±2% of cells versus 13±2% showed RAD51 foci formation, P=0.74). Upon 10μM olaparib exposure, MMR-deficient tumor cultures accumulated less RAD51-positive foci (19±3% in MMR-deficient versus 37±4% in MMR-proficient cells; P=0.02), thereby confirming that these tumors were at least partly deficient in DNA DSB repair by HR. We also investigated the degree of unrepaired DNA damage by H2AX immunofluorescence and olaparib drastically increased the number of H2AX foci in all cells regardless of their MMR status. Furthermore, MMR-deficient cultures exhibited a dose-dependent reduction in proliferation under olaparib, whereas MMR-proficient cells did not respond (P<0.001 by repeated measurement). On average, 1, 3 and 10μM of olaparib decreased proliferation of MMR-deficient cells by respectively 15%, 20% and 42% (P=0.02, P<0.001 and P<0.001, respectively versus untreated cells), whereas no effect was seen in MMR-proficient tumors (P=NS for all concentrations of olaparib). Finally, high-throughput profiling of hotspot mutations also more accurately detected microsatellite instability (MSI) in several cancer types compared to standard diagnostic methods. Sequencing of MMR-deficient tumors thus identified an unexpected synthetically lethal interaction between the major DNA repair pathways, offering novel treatment opportunities for MMR-deficient tumors. Citation Format: Betül T. Yesilyurt, Hui Zhao, Xavier Sagaert, Lieve Coenegrachts, Zeynep Kalender, Diego Garcia, Kim De Keersmaker, Gert Matthijs, Bernard Thienpont, Stein Aerts, Jan Cools, Frederic Amant, Diether Lambrechts. Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2013-2006

The Hemimelic extra toes mouse mutant: Historical perspective on unraveling mechanisms of dysmorphogenesis

Hemimelic extra toes (Hx) arose spontaneously as a dominant mutation in B10.D2/nSnJ mice in 1967. It specifically affects the appendicular skeleton, causing variable foreshortening of the tibia (radius) and preaxial polydactylism. Early anatomical studies revealed anterior overgrowth of the autopod, with decreased apoptosis and increased mitosis in the anterior apical ectodermal ridge and underlying mesenchyme; overextension of apoptosis in the central zeugopod accounted for hemimelia. The Hx mutant phenotype was coarsely mapped to mouse chromosome (Chr) 5 and closely linked to engrailed-2 (En2) and Sonic hedgehog (Shh). This region is syntenic to human Chr 7q36 that harbors several dominant mutations affecting the hand. High-resolution genome mapping identified the Hx mutation as a G --> A base pair transition within Intron 5 of the murine Lmbr1 locus. The critical effect is on a multifunctional conserved regulatory element that acts as a limb-specific, long-distance cis-acting enhancer of Shh expression. As such, the Hx mutant phenotype results from ectopic Shh signals at the anterior margin of the limb bud that directly or indirectly alter FGF4 signaling from the apical ectodermal ridge. Given significant advances in understanding of embryonic development in general and limb development in particular, this review article reveals how research that once attracted interest of teratologists has advanced across the decades to pinpoint a critical molecular lesion and reveal a potential mechanism of a specific malformation that is found commonly in experimental teratology.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

Hydroxylamine-induced purple mutants (ad-3) in Neurospora crassa. II. Identification of genetic alteration at the molecular level.

There is evidence that most hydroxylamine (HA)-induced mutants in Neurospora arise from a GC-to-AT base-pair transition. Evidence supporting this hypothesis was obtained by a study of the revertibility of HA-induced mutants after treatment with specific chemical mutagens. Hydroxylamine-induced ad-3 mutants of Neurospora crassa were tested for revertibility after treatment with nitrous acid (NA), ethyl methanesulfonate (EMS), O-methyl-hydroxylamine (OMHA), and 2-methoxy-6-chloro-9- [3-(ethyl-2-chloraethyl) amino propyl-amino] acridine dihydrochloride (ICR-170). Mutants not reverting after treatment with these mutagens were tested for revertibility after treatment with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). An analysis was made of mutants induced in a 2-component heterokaryon. Of this material 78.2% reverted by an AT-to-GC transition; 10% by a GC-to-AT transition, 4.5% by base-pair insertion or deletion; and 7.3% reverted spontaneously but not after treatment with any of the 5 mutagens. Thus, HA produces mainly GC-to-AT transitions when the treatment is carried out under conditions promoting this specificity. This means that HA has a mutagenic specificity in higher organisms similar to that observed in phage and transforming DNA from B. subtilis. HA can be used as an analytical tool for mutagenesis in higher organisms to characterize the mutagenic activity of agents with an unknown mutation mechanism. HA can also be used to produce mutants with AT at the mutant site. The spectra of genetic alterations among the ad-3B mutants with nonpolarized or polarized complementation patterns and among noncomplementing mutants do not differ from each other, because the majority of HA-induced mutants result from the same genetic alteration.

Association between manganese superoxide dismutase promoter gene polymorphism and breast cancer survival

BackgroundManganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk and survival in breast cancer. This base pair transition (-9 T > C) leads to a valine to alanine amino acid change in the mitochondrial targeting sequence. A polymorphism has also been identified in the proximal region of the promoter (-102 C>T) that alters the recognition sequence of the AP-2 transcription factor, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the -102 C>T polymorphism with overall and relapse-free breast cancer survival in a hospital-based case-only study.Materials and methodsThe relationship between the MnSOD -102 C>T polymorphism and survival was examined in a cohort of 291 women who received chemotherapy and/or radiotherapy for incident breast cancer. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. Patient survival was evaluated according to the MnSOD genotype using Kaplan–Meier survival functions. Hazard ratios were calculated from adjusted Cox proportional hazards modeling. All statistical tests were two-sided.ResultsIn an evaluation of all women, there was a borderline significant reduction in recurrence-free survival with either one or both variant alleles (CT + TT) when compared with patients with wild-type alleles (CC) (odds ratio, 0.65; 95% confidence interval, 0.42–1.01). When the analysis was restricted to patients receiving radiation therapy, there was a significant reduction in relapse-free survival in women who were heterozygous for the MnSOD -102 genotype (relative risk, 0.40; 95% confidence interval, 0.18–0.86). Similarly, when the homozygous and heterozygous variant genotypes were combined, there remained a significant reduction in relapse-free survival in this group (hazard ratio, 0.42; 95% confidence interval, 0.20–0.87).ConclusionThe MnSOD -102 variant allele appears to be associated with an improved recurrence-free survival in all patients, and more dramatically in subjects who received adjuvant radiation therapy.

No apparent association between genetic polymorphisms (−102 C>T) and (−9 T>C) in the human manganese superoxide dismutase gene and gastric cancer 1

Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk. This one base pair transition (-9 T>C) leads to a Val to Ala amino acid change in the mitochondrial targeting sequence. In addition, the MnSOD promoter contains an activator protein-2 (AP-2) binding site that modifies transcription of MnSOD. Mutations have been identified in the proximal region of the promoter in human tumor cell lines. One of these mutations (-102 C>T) has been shown to change the binding pattern of AP-2, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the (-9 T>C) and (-102 C>T) polymorphisms with gastric cancer in a population-based case-control study conducted in Warsaw, Poland. DNA was obtained from a population based case-control study of stomach cancer conducted in Warsaw, Poland, between 1994 and 1996. The MnSOD -9 T>C genotype was determined by PCR-RFLP assay. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. The frequency of the -102 C>T polymorphism was 41% (38/91) in gastric cancer cases and 38% (50/130) in the controls (odds ratio [OR] 1.1, 95% confidence interval [CI] 0.6-2.1). The frequency of the -9 T>C polymorphism was 44% (202/464) in cases and 56% (262/464) in controls (OR 1.1; 95% CI 0.9-1.37). The lack of association was observed in both non-smokers (OR 1.5; 95% CI 0.7-2.34) and smokers (OR 1.1; 95% CI 0.7-1.7). Furthermore, the association was not significant when smokers were segregated by extent of smoking history. The association of the manganese superoxide dismutase polymorphisms at -102 C>T and the -9 T>C were not found to be associated with gastric cancer in a Polish case-control study.

Minireview: A plethora of estrogen receptors in the brain: where will it end?

Until 1996, when estrogen receptor (ER)-beta was discovered, life seemed simple. The gonadal steroid hormone 17 beta-estradiol had one receptor, the ER, a ligand-inducible nuclear transcription factor. ER variants, the result of base pair insertions, transitions, and deletions, as well as alternative splicing, were considered abnormal and a prominent feature of breast cancer. Since then, like many other scientific beliefs, this concept has increased dramatically in complexity, and we are now faced with an ever-increasing array of estrogen-binding proteins, putative ERs, in the brain as well as in the extraneural targets of estrogen. The end is unlikely to be in sight. Some of these putative receptors have been localized to plasma or nuclear membranes, and others to the cytoplasm and/or nucleus. The molecular characteristics of membrane ERs are still in question, and, in most instances, the proteins have not been sequenced or cloned. However, based on transfection and immunohistochemistry, the generally held view, if not dogma, maintains that both nuclear and plasma membrane-associated ERs probably originate from the same gene and transcript that produce the classical intranuclear receptors ER-alpha and ER-beta. However, the physiological relatedness of this observation remains open to question. This review addresses evidence that, in addition to ER-alpha and ER-beta, there exist a variety of non-ER-alpha/non-ER-beta nuclear, cytoplasmic, and plasma membrane ERs in the brain, including G protein-coupled receptors; a novel, developmentally regulated, membrane-associated ER, ER-X; a functional, truncated ER-alpha variant, ER-46; and a putative ER that is immunochemically, structurally, and functionally completely distinct from ER-alpha and ER-beta.

Macrothrombocytopenia and progressive deafness is due to a mutation in MYH9.

In 1992, a family with hereditary macrothrombocytopenia and progressive sensorineural hearing impairment without renal dysfunction was described. Recently, mutations in MYH9, a nonmuscle myosin heavy chain, have been found in several forms of hereditary macrothrombocytopenia. The hereditary macrothrombocytopenia and hearing loss in the previously reported family is due to a mutation in MYH9 gene. Genomic DNA was extracted from the affected proband. Mutation screening of all MYH9 coding exons was carried out using denaturing high-performance liquid chromatography. Abnormal results were followed by direct sequencing of the exon and comparison of the sequence with the normal MYH9 sequence. The results of denaturing high-performance liquid chromatography suggested a potential sequence alteration in exon 30 of MYH9. Direct sequence analysis of this exon in the affected individual identified a G to A single base pair transition at nucleotide 4270 altering codon 1424. This mutations leads to an amino acid change from aspartate (D) to asparagine (N) in the highly conserved coiled-coil domain. A single base pair transition in MYH9, resulting in an amino acid substitution D1424N, is responsible for macrothrombocytopenia and hearing loss in the kindred under study. The presence of hearing impairment and the absence of renal symptoms, as reported in other families with the same mutation MYH9, further highlights the role of genetic background in expression and modification of the affected phenotype.

Thermal Denaturation of Plasmid DNA Observed by Atomic Force Microscopy

(50 ng/ml) Plasmid DNA solutions were heated at different temperatures, and thermal denaturation of plasmid DNA was directly observed by atomic force microscopy (AFM). At room temperature DNA shows some open circular and supercoiled structure on the surface of new cleaved mica. When temperature was increased above 50°C, DNA molecules were partly denatured and the AFM images show globule structure inside the DNA chains. The globule structure resulted from a separation of double-strands into single ones with a subsequently coil-to-globule transition or intrachain recombination of base pairs. Above 80°C DNA appeared as granular structure, which came from complete denaturation and collapse of single strands. At 100°C DNA took place degradation into pieces. AFM studies also show that thermal denaturation of heterogeneous DNA is a multistep process with A–T base pairs separated first and G–C ones latter.

Regional Features of Lattice Corneal Dystrophy Patients in Aichi Prefecture: An Analysis of the TGFBI Gene

To investigate the genetics of patients with lattice corneal dystrophy (LCD) in Aichi Prefecture, the majority of whom originated from the same area of Hazu-gun. We studied twenty patients who visited Nagoya University Hospital from April 1997 to March 2000 and were diagnosed as having LCD. A complete history including a detailed family history was obtained from each of the patients. DNA analysis was performed on each patient with an informed consent. Fourteen patients had an obvious family history, and 7 pedigrees were detected. Four of the 7 families were from Hazu-gun, and Arg 124 Cys mutation in the TGF-beta indused gene human 3(TGFBI) gene was detected in the families. A family from Nishikasugai-gun had a Leu 518 Pro mutation in the same gene. Six patients were considered sporadic without any family history. However, a heterozygous single base-pair transition (leucine to arginine) was detected in codon 527 of the TGFBI gene in 4 out of these 6 patients. Genetic analysis confirmed that LCD observed in patients from Hazu-gun is a type I LCD caused by an Arg 124 Cys mutation in the TGFBI gene. TGFBI gene mutations, other than the Arg 124 Cys, were detected in a family with LCD type I in Aichi Prefecture. Among the autosomal dominant inherited LCDs, there is at times a sporadic pattern because of its late-onset form.

Influence of Ca2+ cations on low pH-induced DNA structural transitions.

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.

Corneal amyloidosis caused by Leu518Pro mutation of βig-h3 gene

AIMTo report a Japanese family diagnosed clinically as having lattice corneal dystrophy type I (LCDI) in which a Leu518Pro mutation in the βig-h3 gene and not the R124C mutation reported...

Leu518Pro mutation of the βig-h3 gene causes lattice corneal dystrophy type I

PURPOSE: To describe a Japanese family with lattice corneal dystrophy type I, which segregates with a novel mutation, Leu518Pro of the βig-h3 gene.METHODS: DNA was extracted from leukocytes in four members (three affected and one unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 12 of the βig-h3 gene was amplified and analyzed with a molecular biologic method. Clinical data were also collected.RESULTS: Three generations of this family have been positively diagnosed with lattice corneal dystrophy, indicating autosomal dominant inheritance. We found a heterozygous point mutation that segregates with the disease phenotype. It was a single base-pair transition (CTG to CCG, Leu to Pro).CONCLUSIONS: Although it is extremely rare compared with the Arg124Cys mutation of the βig-h3 gene, Leu518Pro mutation of the βig-h3also causes lattice corneal dystrophy type I.

Arg124Cys Mutation of the βig-h3 Gene in a Japanese Family With Lattice Corneal Dystrophy Type I

To characterize severe lattice corneal dystrophy, we analyzed the βig-h3 gene, clinical features, histological findings, and genotype-phenotype correlation in an affected Japanese family. Deoxyribonucleic acid was extracted from leukocytes in 16 members (12 affected and 4 unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 4 of the βig-h3 gene was amplified and analyzed using molecular biological methods. Clinical and pathological data were also collected. We found a heterozygous point mutation that causes the disease phenotype. It was a single base-pair transition leading to an amino acid substitution (CGC→TGC, Arg124Cys). The phenotypic variation within families was not recognized. The affected members in the pedigree demonstrated severe visual disturbance in the third decade and required keratoplasty. Histopathological examination revealed amyloid deposits consisting of short and thin amyloid fibers and lattice corneal dystrophy type I. The heterozygous Arg124Cys mutation reported in Caucasian lattice corneal dystrophy caused severe lattice corneal dystrophy consisting of short and thin amyloid fibers in a Japanese family. Based on our study of many members of the family, we are able to construct the natural course of this disorder from its earliest clinical findings through its late manifestations.