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Here we describe 18 new species of the Marattialean genus Danaea: D. alba, D. ampla, D. antioquiana, D. elongata, D. kessleri and D. panamensis (all in D. subg. Danaea); D. dilatata and D. opaca (in D. subg. Arthrodanaea); and D. andina, D. cuspidopsis, D. erosa, D. nasua, D. peruviana, D. polypinna, D. pumila, D. robbinmoranii, D. ubatubensis, and D. velona (in D. subg. Holodanaea). We provide illustrations, maps of geographical distribution, and nomenclatural notes for the new species. We also lectotypify the name D. media and propose that its recent resurrection was a mistake: both D. media and D. elata are synonyms of D. nodosa, which is widespread in Central America and the Greater Antilles; we find that D. pterorachis is a valid name applicable to the Costa Rican material that has been referred to D. media. We consider D. sellowiana and D. nigrescens as distinct species and these names applicable to D. subg. Danaea material from the Brazilian Atlantic Forest and Amazonia, respectively, and we also lectotypify the name D. nigrescens here. We accept D. cuspidata and D. betancurii as species distinct from D. moritziana. Evolutionary relationships and an identification key for the 79 species we currently recognize in Danaea are published in a parallel paper (Keskiniva & al. 2024). Citation: Keskiniva V. & Tuomisto H. 2024: Danaea (Marattiaceae) keeps diversifying, part 1: eighteen new species. – Willdenowia 53: 173–228 Version of record first published online on 22 January 2024. [ABSTRACT FROM AUTHOR]

DNA data are increasingly being used for phylogenetic inference, and taxon delimitation and identification, but scarcely for the formal description of taxa, despite their undisputable merits in taxonomy. The uncertainty regarding the robustness of DNA diagnoses, however, remains a major impediment to their use. We have developed a new program, mold, that identifies diagnostic nucleotide combinations (DNCs) in DNA sequence alignments for selected taxa, which can be used to provide formal diagnoses of these taxa. To test the robustness of DNA diagnoses, we carry out iterated haplotype subsampling for selected query species in published DNA data sets of varying complexity. We quantify the reliability of diagnosis by diagnosing each query subsample and then checking if this diagnosis remains valid against the entire data set. We demonstrate that widely used types of diagnostic DNA characters are often absent for a query taxon or are not sufficiently reliable. We thus propose a new type of DNA diagnosis, termed "redundant DNC" (or rDNC), which takes into account unsampled genetic diversity, and constitutes a much more reliable descriptor of a taxon. mold successfully retrieves rDNCs for all but two species in the analysed data sets, even in those comprising hundreds of species. mold shows unparalleled efficiency in large DNA data sets and is the only available software capable of compiling DNA diagnoses that suit predefined criteria of reliability. [ABSTRACT FROM AUTHOR]

Strawberry is economically important. Anthracnose caused by Colletotrichum spp. has been a serious threat to this crop globally. To detect and distinguish latent Colletotrichum spp. infection on asymptomatic plants at an affordable cost is crucial for this disease control and the sustainability of strawberry industry. Loop-mediated isothermal amplification (LAMP) assay is superior for the higher sensitiveness, efficiency, and lower cost than other methods for pathogen diagnosis. In this study, six previously reported barcodes were evaluated against DNA templates from strawberry fungal pathogens including six Colletotrichum species and five beyond this genus, and further verified in 40 Colletotrichum isolates. Based on the discernibility revealed, a LAMP assay was developed for the diagnosis of Colletotrichum spp. by designing six primers recognizing the conserved regions in β-tubulin 2 gene from 13 Colletotrichum species of C. gloeosporioides and C. acutatum complexes. The specificity and accuracy of LAMP assay was tested against six Colletotrichum species of two complexes, with a detection sensitivity of 100 pg/μL genomic DNA. Current LAMP assay beginning with a 10 min plant lysis enabled a direct and quick diagnosis of Colletotrichum spp. in strawberry within one hour. Followed by PCR using primers specific to ApMat, Marker 2, and Marker 1, this assay could specifically differentiate Colletotrichum species, at least for the dominant species C. fructicola and C. siamense in China, realizing a diagnosis of latent infection at a species level. Current LAMP-PCR combined protocol allows for a sensitive and efficient detection and differentiation of latent Colletotrichum infection on strawberry, which will be useful for disease management and monitoring pathogen population. [ABSTRACT FROM AUTHOR]

Introduction: Several DNA‐based approaches including a reverse dot‐blot hybridization (RDB) have been established for detection of β‐thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β‐thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for β‐thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. Methods: Four recombinant β‐globin plasmids, including a normal sequence and three different mutant panels covering 10 common β‐thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site‐directed mutagenesis. These positive DNA controls were further validated by RDB analysis. Results: We demonstrated the applicability of established positive DNA controls for β‐thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β‐globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant β‐globin plasmids, are required for detecting 10 common β‐thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. Conclusion: The β‐globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of β‐globin plasmid controls with other mutations, which can also be suitable for other DNA‐based approaches. [ABSTRACT FROM AUTHOR]

Cerebrotendinous xanthomatosis is a rare autosomal recessivedisorder of cholesterol and bile acid metabolism associated withthe sterol 27-hydroxylase gene CYP27A1. The disease is characterizedby a varying age of the onset, progressive course, and widerange of the neurological and extraneural symptoms. MRI of thebrain plays an important diagnostic role. Measurement of theserum cholestanol level and DNA analysis are verification methods.Timely started chenodeoxycholic acid replacement therapyenables slowing down the disease progression and, in some cases,preventing neurological disability. References

The problems in the early diagnosis, primary and secondary prevention of family cancer of the breast and/or ovaries are successfully solved within medical genetic counseling at a cancer clinic. Its genetic diagnosis is confirmed, individual risks for breast and/or ovarian cancer are calculated, risk-modifying factors are studied, and treatment, family planning, and childbirth are discussed during clinicogenetic studies.

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.

Background: Minimal residual disease of the peritoneum is challenging for early cancer detection in gastric cancer (GC). Utility of PCR amplification of cancer-derived DNA has been considered feasible due to its molecular stability, however such markers have never been available in GC clinics. We recently discovered cancer-specific methylation of CDO1 gene in GC, and investigated the clinical potential to detect the minimal residual disease. Methods: One hundred and two GC patients were investigated for peritoneal fluid cytology test (CY), and detection level of the promoter DNA methylation of CDO1 gene was assessed by quantitative methylation specific PCR (Q-MSP) in the sediments (DNA CY). Results: (1) CY1 was pathologically confirmed in 8 cases, while DNA CY1 was detected in 18 cases. All 8 CY1 were DNA CY1. (2) DNA CY1 was recognized in 14.3, 25.0, 20.0, and 42.9%, in macroscopic Type II, small type III, large type III, and type IV, respectively, while it was not recognized in Type 0/I/V. (3) DNA CY1 was prognostic relevance in gastric cancer ( p = 0.0004), and its significance was robust among Type III/IV gastric cancer ( p = 0.006 for overall survival and p = 0.0006 for peritoneal recurrence free survival). (4) The peritoneal recurrence was hardly seen in GC patients with potent perioperative chemotherapy among those with DNA CY1. Conclusions: DNA CY1 detected by Q-MSP for CDO1 gene promoter DNA methylation has a great potential to detect minimal residual disease of the peritoneum in GC clinics as a novel DNA marker. [ABSTRACT FROM AUTHOR]

Abstract Paragangliomas are rare tumours of the autonomic nervous system and occur in sporadic and hereditary forms. They are usually benign and have a low mortality. However, they cause significant morbidity related to their mass effect. Genetic predisposition can occur within the familial tumour syndromes multiple endocrine neoplasia type 2 (MEN 2), von Hippel-Lindau (VHL) and neurofibromatosis type 1 (NF-1), or be due to mutations in genes specific to the development of paraganglioma only. Compared to sporadic forms, familial paragangliomas tend to present at a younger age and at multiple sites. Tumours should be diagnosed and resected as early as possible, as it has been shown that morbidity is related to tumour size. This article gives an overview of the current literature on the origin of the different forms of paragangliomas, DNA diagnosis, as well as biochemical and radiological screening guidelines.

Ataxia with oculomotor apraxia (AOA) is a subgroup of the autosomal recessive ataxias with a characteristic oculomotor apraxia: difficulty in transferring and fixing gaze, inability to direct the eyes to a specified side, not due to muscle weakness. Types AOA1 (the APTX gene) and the relatively frequent AOA2 (the SETX gene) are known, along with the recently (2012) described AOA3 (the PIK3R5 gene). Oculomotor apraxia is also characteristic of Louis–Bar ataxia telangiectasia and its variants. DNA diagnosis of AOA1 and AOA2 has been initiated in the Laboratory of Inherited Metabolic Diseases, Medical Genetics Scientific Center. Clinically typical AOA2 in a patient aged 25 years is described – this is the first Russian case confirmed by DNA analysis. The SETX gene was found to contain a previously undescribed mutation, c.2623-2626 del 4, leading to a frameshift mutation, in the heterozygous state; detection of a single mutation with a corresponding clinical picture is sufficient to confirm the diagnosis; the search for the allelic mutation is under way. The diagnosis was made after seven years, though the inherited nature of the disease was proposed from the very beginning. AOA1 was also confirmed in a 15-year-old patient (not investigated clinically by ourselves): two new mutations were found in the compound heterozygotic state; diagnosis of this early form was also delayed. AOA and oculomotor apraxia, which requires specific detection, are probably underestimated in clinical practice. Timely detection and molecular genetic verification would permit effective medical genetic intervention. [ABSTRACT FROM AUTHOR]

The rice cultivar Hikarishinseiki, a semidwarf isogenotype of Koshihikari carrying the Green Revolution sd1 gene, is increasingly grown in both Japan and the United States. Here, we report DNA diagnosis for Hikarishinseiki targeting its Jukkoku-type sd1 locus, which codes for a defective gibberellin 20-oxidase, with a 1 bp substitution in exon 1 (Jukkoku-type GA20ox- 2 mutant allele: Jukkoku_ GA20ox- 2). An allele-specific primer (ASP)-polymerase chain reaction (PCR) with primers SD1F3 and SD1JR gave a PCR product specific to Jukkoku_ GA20ox- 2. In addition, ASP-PCR with primers SD1F3 and SD1NRM (which contains a mismatch at the third nucleotide from the 3′-terminus of SD1NR) gave a PCR product specific to non- Jukkoku_ GA20ox- 2. Multiplex ASP-PCR using SD1F3, VIC dye-labeled SD1JR, and FAM dye-labeled SD1NRM enabled simultaneous codominant detection of Jukkoku_ GA20ox- 2 and non- Jukkoku_ GA20ox- 2 among 188 cultivars. Also, Hikarishinseiki is identifiable by RM253 polymorphism from 11 cultivars carrying Jukkoku_ GA20ox- 2. Taken together, our results establish a methodology for distinguishing Hikarishinseiki. [ABSTRACT FROM AUTHOR]

Due to the variable expression of multiple endocrine neoplasia type 1 (MEN1), it is difficult to predict the course of the disease. However, knowledge about the normal function of the MEN1 gene product, together with the effects of cellular derangement by subsequent genetic events, has increased considerably. At first, the possible existence of a genotype-phenotype correlation is discussed. Thus, mild- and late-onset phenotypes may be distinguished from more malignant phenotypes depending on the character of the primary MEN1 disease gene mutation. Subsequently, tumor-promoting factors such as gender, additional genetic mutations and ecogenetic factors may contribute to the course of the disease. New developments in management are based on the knowledge and experience of the multidisciplinary teams involved. Finally, the metabolic effects of MEN1 mutations in aged patients are discussed. Early identification of predisposition to the disease, together with knowledge about the natural history of specific mutations, risks of additional mutations and periodic clinical monitoring, allow early treatment and may improve life expectancy and quality of life. INSETS: Box 1. Periodic clinical screening.;Box 2. Suggested criteria for MEN1 gene mutation analysis.;Key issues. [ABSTRACT FROM AUTHOR]

Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence. [ABSTRACT FROM PUBLISHER]

Abstract: The rice super cultivar ‘Koshihikari’, grown on 37% of the total rice area in Japan, has been a leading cultivar since 1979. It is also grown in the USA and Australia. However, its tendency to lodge makes harvest difficult and reduces grain quality, and the regularity of large-scale typhoons compromises its production nationally. The genetic improvement of Koshihikari against lodging has been an extremely important theme for the past 30 years in Japan. This paper describes the development of a semidwarf form of Koshihikari by the fixing of an allele of the Green Revolution semidwarf gene sd1 via eight recurrent backcrosses. The cultivar, with excellent flavor and high yield, was named ‘Hikarishinseiki’, which means “Bright New Century” (rice cultivar No. 12273; Ministry of Agriculture, Forestry and Fisheries of Japan: MAFF). Hikarishinseiki, with ≥99.8% of the Koshihikari genome, is about 20cm shorter than Koshihikari. DNA monitoring of Hikarishinseiki confirmed its genomic substitution into Koshihikari except for sd1 and a marker-based method to distinguish Koshihikari from Hikarishinseiki was developed. Its flavor (upper medium grade) and quality were the same as those of Koshihikari grown at 10 sites across Japan, but its yield was on average 3% higher (maximum 29% higher at Hyogo) than that of Koshihikari and it was highly resistant to lodging. Hikarishinseiki is the first semidwarf form of Koshihikari registered by MAFF. Some economical impact has been achieved by introducing Hikarishinseiki and it is already notified as a brand rice description in the rice-producing districts of Niigata, Shiga, Tottori, Okayama, Tokushima, and Kochi prefectures. [Copyright &y& Elsevier]

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominantly inherited tumor syndrome subclassified into three distinct syndromes: MEN 2A, MEN 2B and familial medullary thyroid carcinoma. In MEN 2 families, medullary thyroid carcinoma, pheochromocytomas and parathyroid adenomas occur with a variable frequency, also depending on the specific genetic defect involved. In 1993, the responsible MEN2 gene was identified. The genetic defect in these disorders involves the RET proto-oncogene on chromosome 10. The germline RET mutations result in a gain-of-function of the RET protein. Extensive studies on large families revealed that there is a strong genotype-phenotype correlation. In this review, guidelines for early diagnosis, including MEN2 gene mutation analysis, and treatment, including preventive surgery, periodic and clinical monitoring, have been formulated, enabling improvement of life expectancy and quality of life. Identification of the RET protein has also provided new insights into its function, and the specific pathways it effects involved in cell proliferation, migration, differentiation and survival. In the near future, identification of biological tumor markers will enable target-directed intervention and may prevent and/or delay progression of both primary and residual tumor growth. INSETS: Criteria for DNA-analysis for MEN 2/FMTC.;Key issues. [ABSTRACT FROM AUTHOR]

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal-dominant inherited tumor syndrome characterized by hyperplasia and/or tumors in the parathyroid glands, the pancreatic islets, the anterior pituitary and adrenal glands, as well as neuroendocrine tumors in the thymus, lungs and stomach, and tumors in nonendocrine tissues. In 1997, the responsible MEN1 gene was identified as a tumor-suppressor gene and its product was named menin. In this review, guidelines for early diagnosis, including MEN1 gene mutation analysis, and treatment, including periodic clinical monitoring, have been formulated, enabling improvement of life expectancy and quality of life. Identification of menin-interacting proteins has provided new insights into the function of menin, notably involving regulation of gene transcription related to proliferation and apoptosis, genome stability and DNA repair, and endocrine/metabolic homeostasis. In the near future, target-directed intervention may prevent or delay the onset of MEN 1-related tumors. INSETS: Box 1. Criteria for MEN1 gene mutation analysis.;Box 2. Periodic clinical monitoring.;Key issues. [ABSTRACT FROM AUTHOR]

Abstract: The L1 gene of human papillomavirus-18 (HPV-18) is consistently hypermethylated in cervical carcinomas, but frequently hypo- or unmethylated in exfoliated cells from asymptomatic patients. In precancerous lesions, L1 is sporadically hypermethylated, correlating with the severity of the neoplasia. In order to explore the potential of using L1 methylation as a workable biomarker for carcinogenic progression of HPV-18 infections in routinely taken samples, our aim was to develop methylation–detection techniques that were sensitive and rapid without being overly complex technically. Therein, we developed a methylation-specific PCR (MSP) through the design of primer sets that specifically amplify either methylated or unmethylated HPV-18 L1 DNA within bisulfite-modified sample DNA. Amplification of unmethylated and in vitro methylated HPV-18 DNA by MSP resulted in 2500 copies of either of the two L1 DNA species being detected, a satisfactory sensitivity considering that bisulfite treatment leads to the fragmentation of about 99% of sample DNA. The primers proved specific and did not generate false positive results at concentrations exceeding the lowest limit of detection by a factor of 400. DNA from carcinomas yielded PCR signals only with the methylation-specific primers, and not with primers specific for unmethylated L1 genes. The inverse result was obtained with DNA from precursor lesions that contained only hypomethylated DNA. High-grade precursor lesions and carcinomas that contained hyper- as well as hypomethylated L1 DNA yielded PCR signals with both primers. By developing a fluorescence based real-time PCR, we quantitatively analyzed samples with in vitro methylated and unmethylated L1 DNA, and could distinguish clinical samples with hyper- and hypomethylated DNA or mixtures of both DNAs. The methylation-specific and real-time PCR techniques permitted efficient HPV-18 L1 methylation analyses and open the door for larger-scale clinical studies where the utility of methylation status to predict the progression of HPV-18 infection and HPV-18 associated lesions is assessed. [Copyright &y& Elsevier]

The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes. [ABSTRACT FROM AUTHOR]

Normally, the ability to digest milk sugar (lactose) is present in every child, but not in every adult. The decrease in lactase synthesis (hypolactasia) results in the inability to digest whole milk. Recent studies of the Finnish population have associated lactase persistence in adults with allele T of the C/T−13910 polymorphism located upstream of the lactase gene; a 100% correlation of primary hypolactasia with genotype C/C has been proved. In this study, the allele and genotype frequencies of C/T−13910 were determined in populations of Russia. The frequencies of genotype C/C, varying from 36.6% in Russians to 88.2% in Chukchi, were close to the published medical and epidemiological data on the hypolactasia frequencies in these populations. Genotyping was carried out by three different methods to determine the optimal one. Genotype C/C proved to be the key determinant of primary hypolactasia. It was assumed that DNA diagnosis of genotype C/C provides a predictive test to detect primary hypolactasia long before its clinical manifestation. [ABSTRACT FROM AUTHOR]

Abstract: A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip. [Copyright &y& Elsevier]

Two exotic crane fly species, Tipula (Tipula) paludosa Meigen (Diptera: Tipulidae) and Tipula (Tipula) oleracea L. have spread considerably in North America beyond original areas of detection in eastern and western Canada. These species are endemic in Europe, and pests in pastures and cereals. The two species differ in life cycles and periods when they feed, warranting species-specific control. Identification presents a challenge because the larvae are not easily distinguishable from each other, and resemble native nonpestiferous species present in sympatry. We collected crane flies from urban landscapes and agricultural fields in Oregon in the western United States. Using polymerase chain reaction (PCR), we sequenced a portion of the mitochondrial cytB gene in 55 individuals (eight adults and 47 larvae) from 29 sites. We observed 7% divergence between exotic and native species. Phylogenetic analysis, using Nephrotoma ferruginea F. as an outgroup, resolved four well-supported monophyletic groups: the exotics, T. (Tipula) oleracea and T. (Tipula) paludosa, and two natives, Tipula (Serratipula) tristis Doane and Tipula (Triplicitipula) sp. Nucleotide divergence between T. oleracea and T. paludosa was P = 0.071, whereas within species divergence was very low (P = 0.0018 and P = 0.0022, respectively). The study indicated that mitochondrial cytB sequences provided all accurate, rapid, and economic technique for separation of T oleracea and T. paludosa from each other and from native species, and insights on their habitats. The technique will facilitate early pest management decisions, and studies on host plants and geographic distribution, as the two exotic species extend their ranges across North America. [ABSTRACT FROM AUTHOR]

In the breeding of domestic animals, selection of economically desired traits has been the most important consideration for the improvement of animals, but excluding negative factors in animal production, such as causative genes for hereditary diseases, is also required for the genetic improvement of domestic animals. The incidence of various hereditary diseases has been reported in Japanese beef cattle and these diseases have caused serious problems in the breeding and raising of healthy beef cattle. This article reviews the identification of causative genes for the following three hereditary diseases in Japanese beef cattle: (i) Chediak–Higashi syndrome; (ii) renal tubular dysplasia; and (iii) bovine chondrodysplastic dwarfism. Chediak–Higashi syndrome is a hereditary bleeding disorder reported in Japanese black cattle. To identify the cause of this disease, we cloned and sequenced the bovine LYST gene, which has been found to be involved in Chediak–Higashi syndrome in humans, and found that an amino acid substitution of histidine to arginine at amino acid residue 2015 is the causative mutation in the cattle disease. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation. We mapped the locus for this disease to the 4 cM region of bovine chromosome 1 by linkage analysis and found a large deletion in this region. The deleted region contained the PCLN1 gene encoding a tight-junction protein of renal epithelial cells, and we concluded that deletion of the PCLN1 gene is responsible for the disease. Bovine chondrodysplastic dwarfism is a hereditary disease of Japanese brown cattle, displayed by short limbs and joint abnormalities. We mapped the locus for the disease to a region of bovine chromosome 6 by linkage analysis. By constructing YAC and BAC contigs covering this region and sequence analysis, we identified a novel gene ( LIMBIN), which plays an essential role in bone formation in this region, and found two mutations responsible for the disease. The identification of these mutations provided the basis for DNA-based diagnostic systems for these three diseases, and after development of the diagnosis systems, the incidences of these hereditary diseases have dramatically decreased. [ABSTRACT FROM AUTHOR]

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda–lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 °C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications. [Copyright &y& Elsevier]

A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40 °C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40 °C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40 °C which indicates that the difference of melting temperature (Tm) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of Tm by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (KA) were calculated and as expected, the ratio of KA between the complementary and mismatched analyte was of the order of 102 for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system. [Copyright &y& Elsevier]

The present study was conducted to detect Salmonella typhi by polymerase chain reaction (PCR) in a clinical setting. A group of 40 clinically suspected cases of typhoid fever, lasting for about 3–11 days, with or without chills and rigors and hepatosplenomegaly were selected. Of these, 20 were culture positive and the remaining 20 were found to be negative by conventional blood culture technique. Primary PCR was followed by nested PCR using two sets of primers corresponding to flagellar gene of S. typhi strain. Two bands of about 458 and 343 bp were detected in 20 blood culture positive cases and 12 of the 20 culture negative ones. In the simulated group of samples, no amplification was detected. Our results suggest that PCR-based diagnosis is particularly useful for all clinically suspected cases of typhoid fever. The sensitivity of PCR and its potential use in routine diagnosis and epidemiological studies of typhoid fever can be exploited to complement studies by including bone marrow culture, faeces and bile samples. [Copyright &y& Elsevier]

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by a multitude of low-density lipoprotein (LDL) receptor gene mutations. The LDL receptor is a cell surface trans-membrane protein that mediates the uptake & lysosomal degradation of plasma LDI., thereby providing cholesterol to cells. Affected individuals have elevated plasma levels of LDL, which causes premature coronary atherosclerosis. FH has an estimated worldwide prevalence of 0.2%. In some subpopulations there is an increased frequency of FH and specific LDL receptor mutations are found to be common due to ‘founder gene effect’. Overall, more than 300 naturally occurring LDL receptor mutations have been described. To data upto ten LDL receptor gene mutations have been identified in Indians in South Africa, suggesting increased incidence of FH among Indians. Most mutations have occurred at CpG dinucleotide, a mutational hotspot in human genetic disease. In our study in 25 hypercholesterolemic subjects we have identified two novel insertion mutations in two patients. But the mutations underlying FH are still undefined in the majority of cases. Mutational heterogeneity on the other-hand has complicated disease diagnosis at DNA level. These findings warrant application of a generalized mutation screening method in search for new LDL receptor gene defects. [ABSTRACT FROM AUTHOR]

The fragile X syndrome is a common familial form of mental retardation and is associated with a rare fragile site at Xq27.3 (FRAXA). This disorder has recently been reported to correlate with length variations of restriction genomic DNA fragments which may due to the amplification of (CCG) trinucleotide repeats located at the FRAXA locus. We described here a rapid preparation method of diagnostic DNA probes for the fragile X syndrome by direct enzymatic amplification of human chromosomal DNA. The PstI-assay, which is Southern blot analysis of DNA samples probed by PCR products, was shown to be sensitive method for diagnostic purposes to detect the size variations specific in the fragile X syndrome. [ABSTRACT FROM AUTHOR]

We identified four families in which we suspected the presence of genetic factors predisposing them to cancer. We examined one family with features suggesting Li-Fraumeni syndrome for the presence of a germline p53 mutation in 13 of its members. To detect germline p53 mutations we performed polymerase chain reaction/nonradioisotopic single-strand conformation polymorphism and DNA sequencing analysis on exons 4–9 of the p53 gene. Mutated polymerase chain reaction–restriction fragment length polymorphism analysis was also performed on exon 5 to confirm the mutation identified by the sequencing analysis. A novel germline p53 mutation was identified at codon 133 (ATG→AGG) in exon 5, resulting in the substitution of arginine for methionine, in all four cancer-affected individuals and in three apparently healthy individuals. We also analyzed tumor specimens for additional p53 mutations in the wild-type alleles using the same methods. However, heterozygosity was retained, and no other additional mutations in the wild-type allele were identified in any of the tumor tissues. It is possible that additional mutations in the wild-type allele are not always necessary for the loss of tumor suppressor functions. This study presents serious clinical and ethical problems about the predictive value of identifying germline p53 mutations in presymptomatic carriers. However, accurate predictive testing will be very useful in identifying unaffected individuals who are at increased risk of developing cancer and in detecting cancer at an early stage. [ABSTRACT FROM AUTHOR]

Mutations in hMSH2 and hMLH1 genes were analyzed in patients from 11 Japanese families that had been diagnosed as carrying hereditary nonpolyposis colorectal cancer (HNPCC) by clinical examination. Germ line mutations of hMSH2 gene were identified in 5 independent families in which colorectal (87% of patients), endometrial (30%), ovarian (17%), gastric (14%), and other cancers existed. Five mutations detected between codons 136 and 811 included single-base substitutions (C→T and T→G), a T deletion, and an A insertion, all of which produced stop codons resulting in truncated proteins, and an A→T substitution at splice donor site of exon 5 which resulted in deletion of this exon. Moreover, one HNPCC family was presumed to have germ line mutation of hMSH2 gene because a somatic mutation of hMSH2 gene was detected in a cancer from a patient in this family. In addition to these 11 families already diagnosed with HNPCC, 3 new families with germ line mutations of hMSH2 gene and hMLH1 gene were found through analysis of DNA from patients who had multiple cancers with alteration in microsatellite DNA. These mutations included an AG deletion at codons 877-878 of hMSH2 gene, an AAG deletion at codons 616-618 of hMLH1 gene, and a C→T single-base substitution at codon 217 of hMLH1 gene. Seven of eight germ line mutations found in this study are new mutations that have not been reported previously. In families in which germ line mutations were identified presymptomatic examination was then carried out using polymerase chain reaction single-strand conformation polymorphism analysis of DNA from peripheral blood, and the result was the detection of family members predisposed to HNPCC who did not yet show signs of cancer. These results indicate the value of DNA analysis in the screening and diagnosis of HNPCC patients and families. [ABSTRACT FROM AUTHOR]

Three families segregating for X-linked ichthyosis (XLI) were analysed using the full-length STS cDNA probe and an anonymous polymorphic DNA sequence closely linked to the STS gene. In patients from two of the families, submicroscopic chromosomal deletions could be detected using both the STS and the GMGX9 ( DXS237 locus) probes. Patients in the third family showed the same hybridization pattern as healthy males following molecular hybridization with either of the probes. The results of DNA analysis (indirect geno-type diagnosis) agree well with those based on the arylsulfatase C/β-gal determination and prove the reliability of the biochemical test. Both methods are discussed for carrier detection, prenatal diagnosis, and genetic conselling. [ABSTRACT FROM AUTHOR]

DNA diagnosis of hemophilias has received much attention in recent years, and different methods are presently available to analyze the coagulation factor IX and VIII genes. This review is intended to serve as a brief guide to current and emerging diagnostic strategies. [ABSTRACT FROM AUTHOR]

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder which is known to cause Reye-like syndrome in children and sudden infant death. A point mutation of lysine329-to-glutamic acid329 substitution in the MCAD gene was recently identified as the most common mutation in patients with MCAD deficiency. This mutation is responsible for about 90% of mutant MCAD alleles in Caucasians. Patients with this type of mutation have a variety of symptoms, indicating that the clinical heterogeneity of MCAD deficiency may not be caused entirely by genetic heterogeneity. Screening for the mutation among newborns in England, Australia, and United States of America indicates the prevalence of carriers to be 1 in 40-107, suggesting the high incidence of the mutation. Since presymptomatic diagnosis and appropriate dietary management are important in MCAD deficiency to prevent life-threatening complications, the relatively high incidence of this disorder may warrant population screening. The most common MCAD mutation can now be detected by DNA diagnostic methods using Guthrie cards. This makes it possible to screen a population efficiently for this potentially fatal disorder. [ABSTRACT FROM AUTHOR]