Animals ; Codon, Nonsense ; Disease ; genetics ; Drosophila ; genetics ; metabolism ; Drosophila Proteins ; genetics ; Humans ; Pseudogenes ; Receptors, Odorant ; genetics

CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis.
Adrenal Hyperplasia, Congenital* ; Alleles ; Gene Conversion ; Genotype ; Haplotypes ; Humans ; Incidence ; Korea ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Pseudogenes ; Spectrum Analysis

Pseudogenes, which have long been described as "fossils", play a very important role in eukaryotic genomes. Recently, studies on the so called "junk gene" have attracted more attention. Far from being silent, pseudogenes participate in various biological activities, including being a part in the transcription process, or participating in the formation of small interfering RNA (siRNA) which regulated gene expression by means of the RNA-interference pathway. Recent studies have also shown that pseudogenes regulate tumor suppression through competing for the microRNA (miRNA) with their parent genes. However, a deeper understanding of function analysis of pseudogenes depends on the comprehensive and accurate identification. With the sequencing completion of many genomes and the innovation of bioinformatics tools, efficient and precise identification of pseudogenes have become available in a genome-wide scale. Our review focused particularly on the method of pseudogene identification, the mechanism of its regulatory roles and its potential to be applied in directed evolution. Besides, the promising research direction of pseudogenes was proposed.
Gene Expression Regulation ; Genes, Tumor Suppressor ; physiology ; MicroRNAs ; genetics ; Pseudogenes ; genetics ; physiology ; RNA Interference ; RNA, Small Interfering ; genetics

Many other human species appeared in evolution in the last 6 million years that have not been able to survive to modern times and are broadly known as archaic humans, as opposed to the extant modern humans. It has always been considered fascinating to compare the modern human genome with that of archaic humans to identify modern human-specific sequence variants and figure out those that made modern humans different from their predecessors or cousin species. Neanderthals are the latest humans to become extinct, and many factors made them the best representatives of archaic humans. Even though a number of comparisons have been made sporadically between Neanderthals and modern humans, mostly following a candidate gene approach, the major breakthrough took place with the sequencing of the Neanderthal genome. The initial genome-wide comparison, based on the first draft of the Neanderthal genome, has generated some interesting inferences regarding variations in functional elements that are not shared by the two species and the debated admixture question. However, there are certain other genetic elements that were not included or included at a smaller scale in those studies, and they should be compared comprehensively to better understand the molecular make-up of modern humans and their phenotypic characteristics. Besides briefly discussing the important outcomes of the comparative analyses made so far between modern humans and Neanderthals, we propose that future comparative studies may include retrotransposons, pseudogenes, and conserved non-coding regions, all of which might have played significant roles during the evolution of modern humans.
Biological Evolution ; Genome* ; Genome, Human ; Humans* ; Neanderthals* ; Pseudogenes ; Retroelements

T-cell receptor gamma alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor gamma sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP.
Animals ; Cats ; Continental Population Groups ; Humans ; Immunotherapy ; Models, Animal ; Open Reading Frames ; Prostate ; Prostatic Neoplasms ; Protein Sorting Signals ; Pseudogenes ; Reading Frames ; Receptors, Antigen, T-Cell ; Recombination, Genetic ; Spleen

OBJECTIVETo investigate the expression of PTEN (phosphatase and tension homology deletion on chromosome 10, PTEN) and its pseudogene PTENP1 in acute leukemia (AL) and correlation between them, and to explore the role of PTENP1 on the PTEN expression in AL cells.

METHODSPTEN and PTENP1 mRNA expression were evaluated in bone marrow (BM) samples from 138 newly diagnosed AL patients and 15 healthy controls by quantitative real-time RT-PCR (qRT-PCR). pCDH1-PTENP1 3'UTR-GFP lentivirus vectors were constructed. 293T cells were transfected by calcium phosphate precipitation to produce retrovirus. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP and pCDH1-PTENP1 3'UTR-GFP respectively. The flow cell sorter was used to sort the HL-60 with GFP positively expressed. The mRNA expression of PTEN and PTENP1 was detected by qRT-PCR, the expression of PTEN protein by western blot, and the impact of PTENP13'UTR on the proliferation of HL-60 cells by MTT assay.

RESULTSAML patients showed significantly lower PTEN and PTENP1 mRNA expression in BM compared to healthy controls. Correlation analysis showed that the expression of PTEN and PTENP1 mRNA were positively correlated (P < 0.05). The 108 cases of PTENP1(+) AML were classified according to the prognostic classification of 2011 NCCN Clinical Practice Guidelines in AML, there was no difference among different subgroups. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP (control group) and pCDH1-PTENP1 3'UTR-GFP respectively. Compared with the control group, PTENP1 mRNA level of HL-60 infected with the retroviral vectors expressing pCDH1-PTENP1 3'UTR-GFP increased significantly, and PTEN mRNA level also increased. While the PTEN protein level and the cell growth rate of the PTENP1 3'UTR group didn't change significantly.

CONCLUSIONPTEN and PTENP1 mRNA expression level of BM cells from AL patients is significantly lower. There is a positive correlation between expression of PTEN and PTENP1 mRNA. PTENP1 may regulate the expression of PTEN in mRNA level.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Female ; Gene Expression ; HL-60 Cells ; Humans ; Leukemia ; genetics ; Male ; Middle Aged ; PTEN Phosphohydrolase ; genetics ; Pseudogenes ; genetics ; RNA, Messenger ; genetics ; Transfection ; Young Adult

Congenital adrenal hyperplasia (CAH), an autosomal recessive disorder, is due to deficiency of the enzymes involved in adrenal steroidogenesis. Phenotypic manifestations vary as a result of the degree of glucocorticoid or mineralocorticoid deficiency and androgen excess present. Among Filipinos, the estimated crude incidence of CAH is approximately 1 in 7,000, which is higher than what is reported in most populations. More than 90% of all cases result from a 21-hydroxylase (21-OH) (cytochrome P450c21) enzyme deficiency involving two 21-OH genes, the active gene (CYP21) and a pseudogene (CYP21P). Studies have shown that mutations result from unequal crossover during meiosis which leads to complete deletion of the gene, gene conversion events or to point mutations. To date, there are no published data on the types of mutations present among Filipinos diagnosed with congenital adrenal hyperplasia. The objective of this study is to describe the profile of Filipino patients diagnosed with CAH and to determine the disease-causing alleles in the 21-OH gene of these patients. Using a method of combined differential polymerase chain reaction and amplification created restriction site approach, direct probing for the presence of known mutations in exons 1,3,4,6,7,8 and intron 2 of the CYP21 and CYP21P genes among Filipino patients with CAH was performed. A total of 12 unrelated CAH patients were examined. A majority of these cases had a premature splicing error mutation at nucleotide 656 of intron 2. The determination of the most frequent alleles in our population can facilitate rapid screening for mutations in the 21-OH gene and lead to a definitive diagnosis of CAH.

Human ; Male ; Female ; Steroid 21-hydroxylase ; Adrenal Hyperplasia, Congenital ; Introns ; Glucocorticoids ; Mineralocorticoids ; Alleles ; Pseudogenes ; Rna Splicing ; Nucleotides

OBJECTIVETo detect the genomic deletion mutation in the NEMO gene of a family with incontinentia pigmenti (IP; MIM 308310).

METHODSA pedigree of IP was investigated. By using long PCR, the Delta4-10 deletion in NEMO gene was tested with specific primers In2/JF3R, and Delta4-10 deletion in pseudogene DeltaNEMO was investigated with primers Rev-2/JF3R. NEMO gene of 80 normal controls was also tested.

RESULTSThe deletion of exons 4-10 in both NEMO gene and the pseudogene DeltaNEMO was detected in all the patients in the family, but was not found in the normal individuals in this IP family and 80 unrelated controls.

CONCLUSIONThe study showed that the family with IP, which showed anticipation, was caused by NEMODelta4-10 deletion in the NEMO gene. Long PCR analysis is proven to be an efficient tool for identification of NEMO rearrangements. It could provide useful information for the genetic counseling of the family involved.

Adolescent ; Child ; Electrophoresis ; Exons ; genetics ; Family ; Female ; Humans ; I-kappa B Kinase ; genetics ; Incontinentia Pigmenti ; genetics ; Infant ; Male ; Pseudogenes ; genetics ; Sequence Deletion

BACKGROUND: The prevalent natural killer (NK) cells induce alloreaction against leukemic cells during post-transplant. NK cell alloreactivity depends on the compatibility of killer cell immunoglobulin-like receptors (KIR) epitopes for graft-versus-host disease. Genotypic expressions of inhibitory or activating KIR in patients with acute myelogenous leukemia (AML) and their HLA-matched sibling donors, as a model for Korean KIR haplotype diversity and NK alloreactivity, were investigated. METHODS: Ninety-two patients in complete remission and their 76 HLA-matched sibling donors were enrolled in this study. All the patients were scheduled to receive allogeneic hematopoietic stem cell transplantations (HSCT). KIR PCR-SSP typing was performed for 19 different kinds of KIR genes and pseudogenes. The PCR data representing the KIR genotypes from both the patients and donors were compared. RESULTS: We found 43 Korean KIR haplotypes. Thirty-three variable haplotypes for the AML patients, in addition to 25 haplotypes for the normal HSCT donors, were demonstrated. Of note, the expressions of specific genes such as 2DL2 (P=0.026), 2DS2 (P=0.042), and 2DS4 (P=0.037) revealed remarkable differences between the patients and the normal donors. Korean HLA-identical sibling pairs showed 38% KIR matches in terms of the gene content and allelic polymorphism. Although the KIR gene content was the same between the patients and the donors, 40% of those matched pairs of patients and donors showed allelic polymorphism, specifically in the context of 2DL5 and 2DS4 genes. CONCLUSION: These results indicate that the expressions of donor inhibitory and activating repertoire of KIR genotypes, even in the HLA-matched sibling setting, are unique parameters to be considered when we perform allogeneic sibling HSCT.
Adult* ; Epitopes ; Genotype ; Graft vs Host Disease ; Haplotypes ; Hematopoietic Stem Cells ; Humans ; Killer Cells, Natural ; Leukemia, Myeloid, Acute* ; Polymerase Chain Reaction ; Pseudogenes ; Receptors, KIR* ; Siblings ; Tissue Donors

PURPOSE: This study was undertaken to characterize clinical features of congenital adrenal hyperplasia (CAH) with steroid 21-hydroxylase deficiency, identify appropriate dose of hydrocortisone to normalize endocrine abnormalities, and correlate between clinical phenotype and genotype of the patients. METHODS: Endocrine and molecular evaluations were carried out in 42 patients at Asan Medical Center during the periods of December-1990 through July-2004. Endocrine laboratory study included the assay of plasma 17-hydroxyprogesterone (17-OHP), ACTH, androstenedione, testosterone, renin, and aldosterone by immunoradiometric assay (IRMA). Mutation study was performed by deletion analysis using specific polymerase chain reaction (PCR) for active gene (CYP21A2) with combination of pseudogene (CYP21A1P) primers as well as direct DNA sequencing of active gene. Auxological parameters, endocrine profiles and bone age by Greulich-Pyle method were monitored during treatment with hydrocortisone in 30 patients. Their medical records have been analyzed retrospectively. The patients were categorized as salt wasting type (SW) and simple virilization type (SV) depending on their clinical features. Auxological, endocrinological parameters, dose of hydrocortisone and genotype were statistically compared between these two group using SPSS 11.5 version. RESULTS: Of the 30 patients, 24 patients were SW and 6 patients were SV. In the cases of SW, the boys were 0.17+/-0.08 years old and the girls were 0.08+/-0.08 years old at diagnosis. Initial ratio of bone age to chronologic age (BA/CA ratio) of the boys was 1.01+/-0.49 and of the girls was 2.25+/-2.67. In the cases of SV, the boys were 6.17+/-0.42 years old and the girls were 6.91+/-10.83 years old at diagnosis. Initial BA/CA ratio of the boys was 2.33+/-0.17 and of the girls was 1.64+/-0.80. Serum 17-OHP, ACTH and renin were normalized in all patients by hydrocortisone treatment. The dosage of hydrocortisone needed to normalize endocrinological features was 22.9+/-8.0 mg/m2/day in boys and 18.0+/-4.0 mg/m2/day in girls in the SW group, 12.5+/-3.8 mg/m2/day in boys and 18.4+/-0.7 mg/m2/day in girls in the SV group. Nineteen types of CYP21 mutation was observed in 42 patients. Q318X, R356W, c.293-13 A>G and null mutation were observed in the SW group. CONCLUSION: The age at the diagnosis in SV was older than in SW. In SV, bone age was older than chronologic age at diagnosis. However, finally measured BA/CA ratio and the dosage of hydrocortisone were not different in two clinical types. There was genotype-phenotype correlation in CAH patients with 21-hydroxylase deficiency.
17-alpha-Hydroxyprogesterone ; Adrenal Hyperplasia, Congenital* ; Adrenocorticotropic Hormone ; Aldosterone ; Androstenedione ; Chungcheongnam-do ; Diagnosis ; Female ; Genetic Association Studies ; Genotype* ; Humans ; Hydrocortisone ; Immunoradiometric Assay ; Medical Records ; Phenotype ; Plasma ; Polymerase Chain Reaction ; Pseudogenes ; Renin ; Retrospective Studies ; Sequence Analysis, DNA ; Steroid 21-Hydroxylase* ; Testosterone ; Virilism