Pseudogene Sequences Research Articles

Loop-mediated isothermal amplification of Neisseria gonorrhoeae porA pseudogene: a rapid and reliable method to detect gonorrhea

Background and objectiveGonorrhea is a sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae. Rapid detection is crucial for effective prevention and treatment. This study developed and tested a low-cost effective method for detecting N. gonorrhoeae, especially in developing countries.MethodsDNA from a N. gonorrhoeae standard strain, as well as from 26 genital secretion samples of gonorrhea patients, were isolated and used for loop-mediated isothermal amplification (LAMP) assay, which was conducted using either an automatic real-time PCR analyzer or a water bath. The amplified porA pseudogene sequence was compared with the NCBI database and the LAMP results were compared with that of the traditional culture method for its sensitivity and specificity.ResultsLAMP was able to detect Neisseria DNA at a concentration as low as 1 pg/µL (1 × 103 CFU/mL cells). The LAMP assay results obtained using an automatic real-time PCR analyzer was similar to that of the water bath. Relative to traditional culture, the sensitivity and specificity of the LAMP assay were 94.7 and 85.7%, respectively.ConclusionLAMP was sensitive and reliable for detecting the porA gene of N. gonorrhoeae. It could be used as a rapid, low cost, and effective method for detecting N. gonorrhoeae.

Nuclear mtDNA pseudogenes as a source of new variants of the mtDNA cytochrome b haplotypes: A case study of Siberian rubythroat Luscinia calliope (Muscicapidae, Aves)

Sequence polymorphism of the mitochondrial DNA cytochrome b gene fragment was analyzed in 21 specimens of subspecies Luscinia calliope calliope (Pallas, 1776) and two specimens of L. c. anadyrensis (Portenko, 1939). On sequence chromatograms, in 19 specimens of L. c. calliope, double peaks of heteroplasmy type in the taxon-specific positions were revealed. Moreover, two clone variants were identified. The first variant was the calliope mitochondrial cyt b gene and the second was the nuclear cyt b pseudogene, similar to the mitochondrial haplotype anadyrensis-camtschatkensis. In L. c. anadyrensis, four clone variants, represented by the mitochondrial calliope and anadyrensis-camtschatkensis cyt b genes and nuclear calliope and sachalinensis cyt b pseudogenes, were identified. Some nuclear cyt b pseudogenes were highly similar (98–99%) to the mitochondrial genes of the subspecies L. c. anadyrensis, L. c. camtschatkensis, and L. c. sachalinensis. In the same time, the majority of nuclear pseudogene sequences were characterized by a high level of polymorphism, caused by nonsynonymous substitutions (up to five substitutions per sequence), the presence of indels in some of the clones, and TAA and TGA stop codons. In our opinion, the mitochondrial haplotypes anadyrensis-camtschatkensis and sachalinensis occurred as a result of intergenomic homologous recombination. This finding provides a new insight into the colonization history of the northeastern part of the range by L. calliope, according to which populating the territory of Chukotka, Kamchatka, and Sakhalin took place at different times and along the independent pathways.

Treatment response of pancreatic cancer cells is significantly impaired by tumor associated stromal cells

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosum Wolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.

627. Concerted Evolution of Pseudogenes Facilitated Contiguous Deletion of the Canine Dystrophin and TMEM47 Genes by Homologous Recombination

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder caused by sporadic mutations in the 2.4 Mb dystrophin gene. The large size of the gene is responsible for one of the highest loss-of-function mutation rates in the entire human genome. The majority of dystrophin mutations that cause DMD are deletions that span multiple exons. As the resulting absence of central immunological tolerance has the documented potential to complicate gene therapies in which recombinant dystrophin is expressed, there is an urgent need for well characterized, deletional null animal models for DMD. Preliminary characterization of a naturally occurring mutation in a German shorthaired Pointer (GSHPMD) suggested that the canine dystrophin gene was disrupted by a single, large deletion. Here, we further characterize the GSHPMD deletion and determine the breakpoints to be within short, highly homologous DNA elements spaced over 5.6 Mb apart, suggesting homologous recombination as the most plausible mechanism. The deletion generously flanks the entire dystrophin gene, and additionally encompasses the TMEM47 gene. This is reminiscent of the famous B.B. deletion in the DMD patient whose DNA was used for subtractive hybridization in the cloning of the DMD and nearby CGD loci. Further investigation revealed that the homologous canine DNA elements are ferritin-like pseudogenes, and surprisingly that there are several additional copies present on the X-chromosome. Phylogenomic comparison of the physical map positions of the orthologous pseudogenes in proximity to the dystrophin gene shows a pattern of positional synteny, consistent with the expansion of this pseudogene family prior to the mammalian radiation. Unexpectedly, only in the canine lineage have the pseudogene sequences remained virtually identical to one another, suggesting the existence of a heretofore uncharacterized process of concerted molecular evolution spanning large physical distances. The fully characterized deletional null animal model represents an invaluable resource for the evaluation of the immunological consequences of recombinant dystrophin expression via gene therapy, as it provides the only mammalian system in which the complete absence of dystrophin prevents the development of central tolerance, as would be expected in most cases of DMD. Thus the GSHPMD model can be uniquely leveraged to address a major safety concern as the field pursues strategies for systemic gene delivery in DMD. Of note, published studies have revealed evidence for deleterious immunological responses following administration of vectors expressing recombinant HUMAN dystrophin in both DMD and the Golden Retriever canine model (NEJM 363: 1429-1437 and Mol. Ther. 18:1501-1508).

PiRNAs from Pig Testis Provide Evidence for a Conserved Role of the Piwi Pathway in Post-Transcriptional Gene Regulation in Mammals.

Piwi-interacting (pi-) RNAs guide germline-expressed Piwi proteins in order to suppress the activity of transposable elements (TEs). But notably, the majority of pachytene piRNAs in mammalian testes is not related to TEs. This raises the question of whether the Piwi/piRNA pathway exerts functions beyond TE silencing. Although gene-derived piRNAs were described many times, a possible gene-regulatory function was doubted due to the absence of antisense piRNAs. Here we sequenced and analyzed piRNAs expressed in the adult testis of the pig, as this taxon possesses the full set of mammalian Piwi paralogs while their spermatozoa are marked by an extreme fitness due to selective breeding. We provide an exhaustive characterization of porcine piRNAs and genomic piRNA clusters. Moreover, we reveal that both sense and antisense piRNAs derive from protein-coding genes, while exhibiting features that clearly show that they originate from the Piwi/piRNA-mediated post-transcriptional silencing pathway, commonly referred to as ping-pong cycle. We further show that the majority of identified piRNA clusters in the porcine genome spans exonic sequences of protein-coding genes or pseudogenes, which reveals a mechanism by which primary antisense piRNAs directed against mRNA can be generated. Our data provide evidence that spliced mRNAs, derived from such loci, are not only targeted by piRNAs but are also subject to ping-pong cycle processing. Finally, we demonstrate that homologous genes are targeted and processed by piRNAs in pig, mouse and human. Altogether, this strongly suggests a conserved role for the mammalian Piwi/piRNA pathway in post-transcriptional regulation of protein-coding genes, which did not receive much attention so far.

Nuclear ribosomal ITS functional paralogs resolve the phylogenetic relationships of a late-Miocene radiation cycad Cycas (Cycadaceae).

Cycas is the most widespread and diverse genus among the ancient cycads, but the extant species could be the product of late Miocene rapid radiations. Taxonomic treatments to date for this genus are quite controversial, which makes it difficult to elucidate its evolutionary history. We cloned 161 genomic ITS sequences from 31 species representing all sections of Cycas. The divergent ITS paralogs were examined within each species and identified as putative pseudogenes, recombinants and functional paralogs. Functional paralogs were used to reconstruct phylogenetic relationships with pseudogene sequences as molecular outgroups, since an unambiguous ITS sequence alignment with their closest relatives, the Zamiaceae, is unachievable. A fully resolved and highly supported tree topology was obtained at the section level, with two major clades including six minor clades. The results fully supported the classification scheme proposed by Hill (2004) at the section level, with the minor clades representing his six sections. The two major clades could be recognised as two subgenera. The obtained pattern of phylogenetic relationships, combined with the different seed dispersal capabilities and paleogeography, allowed us to propose a late Miocene rapid radiation of Cycas that might have been promoted by vicariant events associated with the complex topography and orogeny of South China and adjacent regions. In contrast, transoceanic dispersals might have played an important role in the rapid diversification of sect. Cycas, whose members have evolved a spongy layer in their seeds aiding water dispersals.

Implications of hybridization, NUMTs, and overlooked diversity for DNA Barcoding of Eurasian ground squirrels.

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.

Intra-individual ITS polymorphism and hybridization in Pulmonaria obscura Dumort. and Pulmonaria angustifolia L. (Boraginaceae)

Internal transcribed spacer (ITS) is used as a molecular marker in most phylogenetical analyses in Boraginaceae, an unplaced group in the current angiosperm phylogeny. However, there is no knowledge on intra-individual polymorphism of ITS in Boraginaceae. Difficulties in PCR and sequencing of ITS in Pulmonaria species mentioned in the literature may be seen as indirect evidence of intra-individual polymorphism. This study aims to detect intra-individual polymorphism of ITS1–5.8S–ITS2 in rare species Pulmonariaangustifolia L. and common species Pulmonariaobscura Dumort. Cloning of ITS sequences of P. angustifolia, P. obscura and putative hybrid specimens from mixed population showed intra-individual polymorphism of ITS1–5.8S–ITS2 in all specimens, whereas most of them also contained pseudogenic sequences. Intra-individual sequence divergence of ITS1 and ITS2 spacers was similar in the P. angustifolia and mixed population and significantly higher than in P. obscura. P. angustifolia specimens had nucleotide polymorphisms characteristic to both P. angustifolia and P. obscura and part of P. angustifolia sequences clustered into mostly P. obscura clade on the neighbour-joining trees based on ITS1 sequence and ITS2 sequence and structure data. These results suggest a hybrid origin of all P. angustifolia populations studied. Thus, outbreeding depression is a likely explanation for the decline in populations near the northern border of distribution area of rare species P. angustifolia. Hybrid origin of the whole P. angustifolia is also probable; the central population from Poland showed a similar pattern of polymorphism as distribution edge populations.

Eyes underground: Regression of visual protein networks in subterranean mammals

Regressive evolution involves the degeneration of formerly useful structures in a lineage over time, and may be accompanied by the molecular decay of phenotype-specific genes. The mammalian eye has repeatedly undergone degeneration in taxa that occupy dim-light environments including subterranean habitats. Here we assess whether a decrease in the amount of light that reaches the retina is associated with increased regression of retinal genes, whether the phototransduction and visual cycle pathways degrade in a predictable pattern, and if the timing of retinal gene loss is associated with the entrance of mammalian lineages into subterranean environments. Sequence data were obtained from the publically available genomes of the Cape golden mole (Chrysochloris asiatica), naked mole-rat (Heterocephalus glaber) and star-nosed mole (Condylura cristata) for 65 genes associated with phototransduction, the visual cycle, and other retinal functions. Gene sequences were inspected for inactivating mutations and, when present, pseudogene sequences were compared to sequences from subaerial outgroup species. To test whether retinal degeneration is correlated with historical entrances into subterranean environments, estimated dates of retinal gene inactivation were compared to the fossil record and phylogenetic inferences of ancestral fossoriality. Our results show that (1) lower levels of light available to the retina correspond with an increase in the number of retinal pseudogenes, (2) retinal protein networks generally degrade in a predictable manner, although the extensive loss of cone phototransduction genes in Heterocephalus raises further questions regarding SWS1-cone monochromacy versus functional rod monochromacy in this species, and (3) inactivation dates of retinal genes usually post-date inferred entrances into subterranean habitats.

Dealing with pseudogenes in molecular diagnostics in the next-generation sequencing era.

In this chapter, we focus on issues related to the application of next-generation sequencing (NGS) strategies for the analysis of genes with pseudogenes in a clinical setting. Hereby, target enrichment and mapping strategies are crucial factors to avoid "contaminating" pseudogene sequences, which are characterized by higher mutation rates than their functional parental genes. For the target enrichment strategies, we describe advantages and disadvantages of PCR- and capture-based enrichment methodologies. For the mapping strategies, we discuss crucial parameters that need to be considered to accurately distinguish sequences of functional genes from pseudogenic sequences. Finally, we discuss some concrete examples of genes with known pseudogenes and associated with Mendelian disorders that were analyzed by NGS on various platforms and starting from different library preparations.

Discrimination of pseudogene and parental gene DNA methylation using allelic bisulfite sequencing.

Determining the methylation status of genes with pseudogenes can be technically challenging due to sequence homology. High sequence homology can result in the amplification of both pseudogene and parental gene alleles, potentially leading to data misinterpretation. Allelic bisulfite sequencing allows for detection of the methylation status of individual alleles at nucleotide resolution and represents the most reliable method for discriminating pseudogene and parental gene sequences. Here, we discuss important points that should be considered when investigating pseudogene and parental gene methylation status and we describe the method of allelic bisulfite sequencing, including assay design.

Characterization of MHC class II B polymorphism in bottlenecked New Zealand saddlebacks reveals low levels of genetic diversity

The major histocompatibility complex (MHC) is integral to the vertebrate adaptive immune system. Characterizing diversity at functional MHC genes is invaluable for elucidating patterns of adaptive variation in wild populations, and is particularly interesting in species of conservation concern, which may suffer from reduced genetic diversity and compromised disease resilience. Here, we use next generation sequencing to investigate MHC class II B (MHCIIB) diversity in two sister taxa of New Zealand birds: South Island saddleback (SIS), Philesturnus carunculatus, and North Island saddleback (NIS), Philesturnus rufusater. These two species represent a passerine family outside the more extensively studied Passerida infraorder, and both have experienced historic bottlenecks. We examined exon 2 sequence data from populations that represent the majority of genetic diversity remaining in each species. A high level of locus co-amplification was detected, with from 1 to 4 and 3 to 12 putative alleles per individual for South and North Island birds, respectively. We found strong evidence for historic balancing selection in peptide-binding regions of putative alleles, and we identified a cluster combining non-classical loci and pseudogene sequences from both species, although no sequences were shared between the species. Fewer total alleles and fewer alleles per bird in SIS may be a consequence of their more severe bottleneck history; however, overall nucleotide diversity was similar between the species. Our characterization of MHCIIB diversity in two closely related species of New Zealand saddlebacks provides an important step in understanding the mechanisms shaping MHC diversity in wild, bottlenecked populations.

The B-hordein prolamin family of barley

The spectrum of B-hordein prolamins and genes in the single barley cultivar Barke is described from an in silico analysis of 1452 B-hordein ESTs and available genomic DNA. Eleven unique B-hordein proteins are derived from EST contigs. Ten contigs encode apparent full-length B-hordeins and the eleventh contains a premature stop codon that will lead to a truncated B-hordein. The 11 sequences are placed within the two previously described classes, i.e., the B1- and B3-type B-hordeins. The number of ESTs assigned to each sequence is used as an estimate of relative gene transcription and expression. Three of the sequences account for 79% of the total ESTs, with one sequence comprises 32% of the total ESTs and has a variant C-terminus caused by an undefined sequence change history near the 3' coding terminus. The 70× difference in EST distribution among sequences points to the importance of understanding differential rates of expression within closely related gene families. Analysis of available genomic sequences confirms the EST assembly and reveals one full-length and two partial sequences of pseudogenes as evidenced by no matching ESTs for the sequences and premature stop codons and frame shifts.

Ribosomal protein SA and its pseudogenes in ruminants: an extremely conserved gene family

The ribosomal protein SA (RPSA), also known as 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR), has been identified as a multifunctional protein, playing an important role in multiple pathologies like cancer and prion diseases. Since RPSA is involved in the binding and internalization of the prion protein, mutations in the ovine RPSA gene, influencing the RPSA-PrP<sup>C</sup>/PrP<sup>Sc </sup>binding, can potentially play a part in the resistance to prion diseases. Our goal was to further characterize the complex RPSA gene family and to detect structural mutations which can play a role in this disease. In a prior study, 11 ovine pseudogenes were detected experimentally. As the whole genome shotgun ovine genome became accessible, an in silico genome-wide screening was performed and 37 new pseudogenes (36 processed and one semi-processed pseudogene) were detected, bringing the total to 48 ovine RPSA pseudogenes. Additionally, the complete bovine genome was screened in silico and 56 pseudogenes were identified. Once these sequences were known, it was possible to analyze the presence of mutations in the coding sequence and exon-flanking regions of the ovine functional full-length RPSA gene without the interference of pseudogenic sequences. Nineteen mutations were found: one in the 5’ UTR, a silent one in the coding region, and seventeen in the exon-flanking regions, including an interesting mutation in the SNORA62 gene, localized in intron 4 of RPSA, leading to potential ribosomal defects. Structural mutations of the RPSA gene can be ruled out to play a role in transmissible spongiform encephalopathies but regulatory mutations still can have an effect on these diseases.

Detection of large scale 3′ deletions in the PMS2 gene amongst Colon-CFR participants: have we been missing anything?

Current screening practices have been able to identify PMS2 mutations in 78 % of cases of colorectal cancer from the Colorectal Cancer Family Registry (Colon CFR) which showed solitary loss of the PMS2 protein. However the detection of large-scale deletions in the 3' end of the PMS2 gene has not been possible due to technical difficulties associated with pseudogene sequences. Here, we utilised a recently described MLPA/long-range PCR-based approach to screen the remaining 22 % (n = 16) of CRC-affected probands for mutations in the 3' end of the PMS2 gene. No deletions encompassing any or all of exons 12 through 15 were identified; therefore, our results suggest that 3' deletions in PMS2 are not a frequent occurrence in such families.

The Ly49 Gene Family. A Brief Guide to the Nomenclature, Genetics, and Role in Intracellular Infection

Understanding the Ly49 gene family can be challenging in terms of nomenclature and genetic organization. The Ly49 gene family has two major gene nomenclature systems, Ly49 and Killer Cell Lectin-like Receptor subfamily A (klra). Mice from different strains have varying numbers of these genes with strain specific allelic variants, duplications, deletions, and pseudogene sequences. Some members activate NK lymphocytes, invariant NKT (iNKT) lymphocytes and γδ T lymphocytes while others inhibit killing activity. One family member, Ly49Q, is expressed only on myeloid cells and is not found on NK, iNKT, or γδ T cells. There is growing evidence that these receptors may regulate not just the immune response to viruses, but other intracellular pathogens as well. Thus, this review’s primary goal is to provide a guide for researchers first encountering the Ly49 gene family and a foundation for future studies on the role that these gene products play in the immune response, particularly the response to intracellular viral and bacterial pathogens.

Evolutionary Genetics of the Hydroid Allodeterminant alr2

We surveyed genetic variation in alr2, an allodeterminant of the colonial hydroid Hydractinia symbiolongicarpus. We generated cDNA from a sample of 239 Hydractinia colonies collected at Lighthouse Point, Connecticut, and identified 473 alr2 alleles, 198 of which were unique. Rarefaction analysis suggested that the sample was near saturation. Most alleles were rare, with 86% occurring at frequencies of 1% or less. Alleles were highly variable, diverging on average by 18% of the amino acids in a predicted extracellular domain of the molecule. Analysis of 152 full-length alleles confirmed the existence of two structural types, defined by exons 4-8 of the gene. Several residues of the predicted immunoglobulin superfamily-like domains display signatures of positive selection. We also identified 77 unique alr2 pseudogene sequences from 85 colonies. Twenty-seven of these sequences matched expressed alr2 sequences from other colonies. This observation is consistent with pseudogenes contributing to alr2 diversification through sequence donation. A more limited collection of animals was made from a distant, relict population of H. symbiolongicarpus. Sixty percent of the unique sequences identified in this sample were found to match sequences from the Lighthouse Point population. The large number of alr2 alleles, their degree of divergence, the predominance of rare alleles in the population, their persistence over broad spatial and temporal scales, and the signatures of positive selection in multiple residues of the putative recognition domain paint a consistent picture of negative-frequency-dependent selection operating in this system. The genetic diversity observed at alr2 is comparable to that of the most highly polymorphic genetic systems known to date.

Comparative Analysis of a Putative Tuberculosis-Susceptibility Gene, MC3R, and Pseudogene Sequences in Cattle, African Buffalo, Hyena, Rhinoceros and Other African Bovids and Ruminants

Studies in humans have suggested the possible involvement of melanocortin-3-receptor (MC3R) and other components of the central melanocortin system in host defense against mycobacteria. We report a genomic DNA nucleotide sequence highly homologous to human MC3R in several bovids and non-bovid African wildlife species. Nucleotide sequence analysis indicates that the orthologous genes of cattle and buffalo are highly homologous (89.4 and 90%, respectively) to the human MC3R gene. Sequence results also identified a typical non-functional, duplicated pseudogene, MC3RP, in 7 species from the family Bovidae. No pseudogene was found in animals outside Bovidae. The presence of the pseudogene in tuberculosis-susceptible species could have possible immunomodulatory effects on susceptibility to bovine tuberculosis infection, as well as a considerable influence on energy metabolism and food conversion efficiency.

A PIK3CA Pyrosequencing-Based Assay that Excludes Pseudogene Interference

Phosphatidylinositol 3'-kinase gene (PIK3CA) encodes a lipid kinase that regulates signaling pathways downstream of epidermal growth factor receptor and is mutated in 10% to 30% of colorectal cancers. Activating mutations in this gene up-regulates the AKT signaling pathway, making it a potentially useful therapeutic target. Mutations in PIK3CA are not exclusive of mutations in KRAS, BRAF, or NRAS. We designed a pyrosequencing assay to detect mutations in all three positions of codons 542 and 545 in exon 9 and codon 1047 in exon 20 of this gene. The exon 9 reverse PCR primer was designed to avoid amplifying a pseudogene in chromosome 22 that has >95% homology with exons 9 through 13 in PIK3CA. Two hundred colorectal cancers from FFPE tissue previously characterized for KRAS mutation status were evaluated for PIK3CA mutations. In KRAS-mutated samples, 20% had an additional mutation in PIK3CA. The mutation rate in KRAS wild-type samples was 7.5%. When using our assay, pseudogene was not observed in any of these samples. In addition, pseudogene- and gene-specific amplification was performed on DNA from 40 additional colorectal cancers. Sequencing of these PCR products yielded the expected gene or pseudogene sequence in all cases. Thus, we have developed a PIK3CA pyrosequencing assay capable of detecting mutations in all three positions in the three hot spot codons with no pseudogene interference.

Identification of a novel recombinant mutation in Korean patients with Gaucher disease using a long-range PCR approach

Gaucher disease (GD) is an autosomal recessive, lysosomal disorder caused by mutations in the gene for the β-glucocerebrosidase (GBA) enzyme. Presence of the non-functional GBAP pseudogene, which shares high sequence similarity with the functional GBA gene, has made it difficult to carry out molecular analyses of GD, especially recombinant mutations. Using a long-range PCR approach that has been skillfully devised for the easy detection of GBA recombinant mutations, we identified four recombinant mutations including two gene conversion alleles, Rec 1a and Rec 8a, one reciprocal gene fusion allele, Rec 1b, and one reciprocal gene duplication allele, Rec 7b, in Korean patients with GD. Rec 8a, in which the GBAP pseudogene sequence from intron 5 to exon 11 is substituted for the GBA gene is a novel recombinant mutation. All mutations were confirmed by full sequencing of PCR amplicons and/or Southern blot analysis. These results indicate that the usage of long-range PCR may allow the rapid and accurate detection of GBA recombinant mutations and contribute to the improvement of genotyping efficiency in GD patients.