Genome Sequencing of Human Cytomegalovirus Strain LCW and Identification of Asia-specific Cluster

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Molecular genomic studies of HCMV have been attempted in order to understand the pathogenesis and evolution of HCMV. However, studies on HCMV strains of Asian origin are limited. In this study, it was attempted to understand the genomics of HCMV isolated from Korea. Clinical strain LCW isolated from Korean patient was passaged in vitro cell culture, and subjected to next-generation sequencing. Complete genome sequence was obtained and compared with other HCMV strains. The LCW genome was found to contain 170 open reading frames (ORFs) and two ORF (RL5A and RL13) of the strain LCW were found to be truncated due to early stop codon. Phylogenetic analysis suggested that the strain LCW was closely related with Asian strains such as HCMV strains JHC and HAN. Common nucleotide sequences among the 3 Asian strains distinguishable from other strains were detected at 197 sites including 104 sites in ORFs.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

A Novel Germline Mutation of the APC Gene: A Case Report of Familial Adenomatous Polyposis Requiring Repeated Endoscopic Resections for Gastroduodenal Polyps

Familial adenomatous polyposis (FAP) is a precancerous clinical entity, which is characterized by the development of numerous adenomatous polyps throughout the colon and rectum. The majority of FAP are associated with mutations of the adenomatous polyposis coli (APC) gene. Until now, more than 1,000 different APC mutations have been reported and some mutations express attenuated phenotypes which are milder forms with 10~100 colorectal polyps. We identified a novel mutation of APC gene which expressed an attenuated FAP but caused large gastroduodenal tubular adenomas requiring repeated endoscopic resections. A 16-year-old girl was referred to Incheon St. Mary's Hospital for evaluation of gastric polyposis. Initial esophagogastroduodenoscopy (EGD) showed numerous gastric polyps in the fundus and upper body and a few polyps in the duodenum. Pathologic examination confirmed gastric polyps as fundic gland polyps and duodenal polyps as tubular adenomas. Only a few colonic polyps of 2 to 5 mm in size were found on colonoscopy. Genetic analysis using polymerase chain reaction and direct sequencing revealed a novel stop codon mutation at codon 1522 in exon 16 of APC gene. At 12-month, 18-month, and 35-month follow-up EGD, large duodenal polyp and gastric polyps were removed endoscopically.

Identification of a novel mutation in a patient with pseudohypoparathyroidism type Ia / 소아과

Pseudohypoparathyroidism type Ia (PHP Ia) is a disorder characterized by multiform hormonal resistance including parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO). It is caused by heterozygous inactivating mutations within the Gs alpha-encoding GNAS exons. A 9-year-old boy presented with clinical and laboratory abnormalities including hypocalcemia, hyperphosphatemia, PTH resistance, multihormone resistance and AHO (round face, short stature, obesity, brachydactyly and osteoma cutis) which were typical of PHP Ia. He had a history of repeated convulsive episodes that started from the age of 2 months. A cranial computed tomography scan showed bilateral calcifications in the basal ganglia and his intelligence quotient testing indicated mild mental retardation. Family history revealed that the patient's maternal relatives, including his grandmother and 2 of his mother's siblings, had features suggestive of AHO. Sequencing of the GNAS gene of the patient identified a heterozygous nonsense mutation within exon 11 (c.637 C>T). The C>T transversion results in an amino acid substitution from Gln to stop codon at codon 213 (p.Gln213*). To our knowledge, this is a novel mutation in GNAS.

Portal-Splenic-Mesenteric Venous Thrombosis in a Patients with Protein S Deficiency due to Novel PROS1 Gene Mutation / 대한소화기학회지

Protein S (PS), a vitamin K-dependent glycoprotein, performs an important role in the anticoagulation cascade as a cofactor of protein C. Because of the presence of a pseudogene and two different forms of PS in the plasma, protein S deficiency (PSD) is one of the most difficult thrombophilias to study and a rare blood disorder associated with an increased risk of thrombosis. We describe a unusual case of previously healthy 37-year-old man diagnosed with portal-splenic-mesenteric vein thrombosis secondary to PSD. The patient was admitted to the hospital due to continuous nonspecific abdominal pain and nausea. Abdominal computed tomography revealed acute venous thrombosis from inferior mesenteric vein to left portal vein via splenic vein, and laboratory test revealed decreased PS antigen level and PS functional activity. Conventional polymerase chain reaction and direct DNA sequencing analysis of the PROS1 gene demonstrated duplication of the 166th base in exon 2 resulting in frame-shift mutation (p.Arg56Lysfs*10) which is the first description of the new PROS1 gene mutation to our knowledge. Results from other studies suggest that the inherited PSD due to a PROS1 gene mutation may cause venous thrombosis in a healthy young man without any known predisposing factor.

The KIT Exon 11 Stop Codon Mutation in Gastrointestinal Stromal Tumors: What Is the Clinical Meaning?

BACKGROUND/AIMS: Gastrointestinal stromal tumors (GISTs) strongly express a receptor tyrosine kinase (RTK, c-KIT-CD117) harboring a KIT mutation that causes constitutive receptor activation leading to the development and growth of tumors; 35% of GISTs without KIT mutations have platelet-derived growth factor receptor alpha (PDGFRA) mutations, and the type of mutation plays an important role in the response to treatment. This study aimed to establish the frequency of stop codon mutations in the RTKs, KIT, and PDGFRA, in GISTs and correlate this molecular alteration with protein expression and treatment responsiveness. METHODS: Seventy-nine GISTs were analyzed for both KIT and PDGFRA mutations. Immunohistochemical expression was studied in tissue microarray blocks. RESULTS: We found three rare KIT mutations in exon 11 that induced a stop codon, two at position 563 and one at position 589, which have never been described before. All three tumors were CD117-, DOG1-, and CD34-positive. Two patients with a KIT stop codon mutation did not respond to imatinib therapy and died shortly after treatment. CONCLUSIONS: The association between stop codon mutations in KIT and patient survival, if confirmed in a larger population, may be useful in choosing effective therapies.

A Novel Germline Mutation in Exon 15 of the APC Gene in Attenuated Familial Adenomatous Polyposis: A Report of Two Cases

Attenuated familial adenomatous polyposis (AFAP) is a variant of familial adenomatous polyposis with fewer than one hundred colorectal polyps and a later age of onset of the cancer. Here, we report two cases of AFAP within family members. Each patient demonstrated the same novel germ line mutation in exon 15 of the adenomatous polyposis coli (APC) gene and was successfully managed with sulindac after refusal to perform colectomy: a 23-year-old man with incidentally diagnosed gastric adenoma and fundic gland polyps underwent colonoscopy, and fewer than 100 colorectal polyps were found; a 48-year-old woman who happened to be the mother of the 23-year-old man also showed fewer than 100 colorectal polyps on colonoscopy. Genetic analysis revealed a novel frameshift mutation in exon 15 of the APC gene. The deletion of adenine-guanine with the insertion of thymine in c.3833-3834 resulted in the formation of stop codon 1,287 in both patients. The patients were treated with sulindac due to their refusal to undergo colectomy. The annual follow-up upper endoscopy and colonoscopy in the following 2 years revealed significant regression of the colorectal polyps in both patients.

Read-through Mutation in the Coat Protein ORF Suppresses Turnip Yellow Mosaic Virus Subgenomic RNA Accumulation

We have previously observed that a sequence in coat protein (CP) ORF of Turnip yellow mosaic virus (TYMV) is required for efficient replication of the virus. The sequence was predicted to take a stem-loop structure, thus termed SL2. While examining various SL2 mutants, we observed that all the modifications resulting in extension of translation beyond the CP ORF significantly suppressed subgenomic RNA accumulation. The genomic RNA level, in contrast, was not affected. Introduction of an in-frame stop codon in the CP ORF of these constructs restored the level of subgenomic RNA. Overall, the results suggest that the read-through makes the subgenomic RNA unstable.

Full sequence analysis for a null allele of MICA gene (MICA*063N) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the full nucleotide sequence of a null allele of major histocompatibility complex class I chain-related gene (MICA).</p><p><b>METHODS</b>A sequence-based typing method was used to determine the nucleotide sequence of the MICA gene. Potential alleles were identified with a computer program.</p><p><b>RESULTS</b>The identified allele has possessed a sequence similar to that of MICA*027 except for a C→T substitution at position 184 in codon 62 (CAG→TAG) of exon 2. As a stop codon, this may result in a truncated protein.</p><p><b>CONCLUSION</b>A null allele of MICA gene has been identified. The sequence has been submitted to the Genbank nucleotide sequence database (submission No. HWS10011131), which was officially named as MICA*063N by the WHO Nomenclature Committee in October 2010.</p>

Cornelia de Lange Syndrome with NIPBL Gene Mutation: A Case Report

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly characterized by distinctive facial features, upper limb malformations, growth and cognitive retardation. The diagnosis of the syndrome is based on the distinctive clinical features. The etiology is still not clear. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A (also called SMC1L1) and SMC3 have been suggested as probable cause of this syndrome. We experienced a case of newborn with CdLS showing bushy eyebrows and synophrys, long curly eyelashes, long philtrum, downturned angles of the mouth and thin upper lips, cleft palate, micrognathia, excessive body hair, micromelia of both hands, flexion contracture of elbows and hypertonicity. We detected a NIPBL gene mutation in a present neonate with CdLS, the first report in Korea.

Localization of polypeptides release factors and ribosome protein L11 in Euplotes octocarinatus / 生物工程学报

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.

Identification of known and novel PTCH mutations in both syndromic and non-syndromic keratocystic odontogenic tumors / 国际口腔科学杂志·英文版

<p><b>AIM</b>To clarify the role of PTCH in patients with NBCCS-related and non-sydromic keratocystic odontogenic tumors.</p><p><b>METHODOLOGY</b>Mutation analysis was undertaken in 8 sporadic and 4 NBCCS-associated KCOTs.</p><p><b>RESULTS</b>Four novel and two known mutations were identified in 2 sporadic and 3 syndromic cases, two of which being germline mutations (c.2179delT, c.2824delC) and 4 somatic mutations (c.3162dupG, c.1362-1374dup, c.1012 C>T, c.403C>T).</p><p><b>CONCLUSION</b>Our findings suggest that defects of PTCH are associated with the pathogenesis of syndromic as well as a subset of non-syndromic KCOTs.</p>

A Case of Tay-Sachs Disease in Korea Diagnosed by Enzyme Assay and DNA Analysis / 소아과

Tay-Sachs disease is an autosomal recessive, neurodegenerative disorder that results from excessive storage of the cell membrane glycolipid, and GM2 ganglioside within the lysosomes of cells. This disease is caused by deficiency of the isoenzyme beta-hexosaminidase A, produced in the endoplasmic reticulum. Patients with Tay-Sachs disease are characterized by normal motor development in the first few months of life, followed by progressive weakness and loss of motor skills beginning around 2 to 6 months of life. Neurodegeneration is relentless, with death occurring by the age of 4 or 5 years. Tay-Sachs disease could be diagnosed by hexosaminidase enzyme assay and DNA analysis of HEXA gene. However, specific treatment has not been developed. We report here on a case of Tay- Sachs disease in 18-month-old male who presented with delayed development and seizure. This patient showed hyperacusis and cherry red spot in macula on examination of the fundus. The hexosaminidase A activity was zero percent in the enzymatic assay and DNA analysis identified a mutation that glutamine is substituted by stop codon at position 390(Q390X). This patient is the first case of Tay-Sachs disease in Korea diagnosed by enzymatic assay and DNA analysis.

X-linked hyper IgM syndrome: a report of the first case in Thailand with a confirmed mutation of CD40 ligand gene.

X-linked hyper IgM (XHIM) syndrome is a rare congenital immunodeficiency disease caused by failure of B cell to isotype switch from IgM to other classes of immunoglobulins in response to infections. Recently, a molecular cloning of the gene responsible for the syndrome, the CD40L gene has been accomplished and the gene was successfully mapped to the long arm of X chromosome at the position Xq26. We, herein, report the first case of molecular proven XHIM in a Thai boy with a classic presentation and with a confirmed mutation of the CD40L gene. Case Report: A.S. was a 1 year 7 month old boy referred from Buriram Provincial Hospital for a work up and treatment for his recurrent infections consisted of chronic respiratory tract infections with otitis media (since 6 months of age), chronic diarrhea (since 9 months of age) and malnutrition (marasmus) secondary to his longstanding illnesses. He was a product of a consanguineous marriage but without history of similar illness observed in his pedigree. Abnormal laboratory works up included IgG of 300 mg/dl, IgA 10 mg/dl, IgM 1,635 mg/dl, positive stool examinations for Cryptosporidium, chronic colitis on radiographic gastrointestinal follow through study, a positive acid fast bacillus (AFB) stain of gastric aspirate and multiple positive bacterial cultures from various body sources. His anti-HIV serology was negative. His hospital course was significant for several bouts of infections of gastrointestinal, respiratory, and genitourinary systems. His treatment consisted of multiple courses of antibiotics, antituberculous drugs and IVIG administrations. His hospital course was complicated with feeding problem from an esophageal stricture requiring several esophageal dilatations. The analysis of CD40L gene revealed a point mutation of exon 5 (A619T) of the CD40L gene resulting in a stop codon confirming that indeed he had XHIM. He died with Pseudomonas septicemia during the waiting period for a bone marrow transplantation from a cord-blood stem cell.

A Molecular Genetic Study with EcoRII Restriction Enzyme on the Steroidogenic Acute Regulatory Protein (StAR) Gene / 대한소아내분비학회지

The molecular defect of congenital lipoid adrenal hyperplasia has been discovered to be in the transport of cholesterol into mitochondria due to defective regulatory protein called "Steroidogenic Acute Regulatory Protein (StAR)", while the enzyme P450scc itself is normal. This study with EcoRII restriction enzyme aimed at elucidating more conveniently the molecular defect in the StAR gene. The genomic DNAs were extracted from their peripheral blood. We amplified the exon 7, hot spot, of the StAR gene with 1 set of primers by Polymerase Chain Reaction (PCR). Subsequently, a PCR product corresponding to target sequence (~437 bps) from the patient and her father have been sequenced by automatic sequence analyzer. The PCR-RFLP (Restriction Fragment Length Polymorphism) analysis after restriction digestion with EcoRII restriction enzyme was also performed on 12% polyacrylamide gel electrophoresis. The mutation was identified in the exon 7 of the StAR gene, substituting C for T at codon 258, consequently replacing glutamine by stop codon. This mutation alters EcoRII restriction site. In addition, we obtained the good result of PCR-RFLP (Restriction Fragment Length Polymorphism) analysis on 12% polyacrylamide gel electrophoresis. Therefore, the PCR-RFLP (Restriction Fragment Length Polymorphism) analysis with EcoRII restriction enzyme can be easily utilized to screen carrier, diagnose the patient prenatally or postnatally.

A Study on Mutations of p53 Tumor Suppressor Gene in Oral Tumors

Nowadays, there are a lot of evidence that mutation of the p53 tumor suppressor gene is one of the most common genetic abnormalities in neoplastic progression. In this study, we analyzed 20 specimens of oral tumors (squamous cell carcinoma 14 cases, ameloblastoma 3 cases, adenoid cystic carcinoma 2 cases, malignant schwannoma 1 case) using polymerase chain reaction and direct sequencing which used an automated DNA sequencer and software for detection of mutations. Polymerase chain reactions were performed with 4 sets of primers encompassing exon 5, 6, 7, 8, and direct sequencing method was employed. The results were as followings. 1. We detected 10 piont mutations out of 20 specimens (50%). 2. The genetic alterations included 7 mis-sense mutations resulting in single amino acid subtitutions, 2 silent mutations, 1 non-sense mutations encoding a stop codon. 3. Mutations were mostly in exon 7(7 out of 10 mutations, 70%) and involved codons 225, 234, 235, 236, 238, 247. 4. Therse were 4 cases of T-->A transversion, 2 cases of C-->A transversion, A-->G transition, 1 case of C-->G, T-->G transversion respectively. 5. We could find out point mutations more conveniently using PCR-Automated Direct Sequencing method.

Mutation at exon 10 of the fibroblast growth factor receptor 3 (FGFR3) in a fetus with thanatophoric dysplasia type I (TDI) / 대한산부인과학회잡지

Thanatophoric dysplasia (TD) is a sporadic lethal type of skeletal dysplasia featuring micromelia, decreased thoracic dimension and macrocephaly. To date, several kinds of mutation in fibroblast growth factor receptor 3 (FGFR3) has been identified in TD. We experienced a case of TD type I and underwent sequencing of the exon 7, 10 and the stop codon of FGFR3 to identify the type of mutation. TDI was diagnosed by the prenatal ultrasound at 25 weeks of gestation. The pregnancy was terminated and the diagnosis was confirmed by radiological and histologic examinations. The genomic DNA was extracted and the sequences of the exon 7, 10 and the stop codon of FGFR3 were amplified by PCR. The sequencing was performed for the each PCR products by dideoxyterminator method. The nucleotide transition from G to T was found in the nucleotide 1108, which is a part of the transmembrane domain, exon 10. To date, only one type of mutation (nucleotide 742) in the FGFR3 was identified in TD1 among Asian. This case firstly reveals the mutation of FGFR3 other than mutation at nucleotide 742 in TD1.

Poliquistosis renal autosomica dominante: deteccion de una nueva mutacion en el gen PKD1 / Autosomal dominant polycystic kidney disease: detection of a novel mutation in the PKD1 gene

La poliquistosis renal autosómica (ADPKD) es una enfermedad hereditaria que presenta heterogeneidad genética. Al menos tres genes son responsables del desarrollo de la enfermedad: PKD1 en el cromosoma 16p 13.3, PKD2 en 4q21 y PKD3, aún no localizado. A partir de la descripción de la secuencia del gen PKD1, el interés general se volcó a la búsqueda de mutaciones causantes de la enfermedad. La mayoría de las mutaciones halladas es de diverso origen y localiza a lo largo del gen, no pudiendo hallarse correlación fenotípica alguna. En este trabajo se describe el hallazgo de una mutación en el exón 44 del gen PKD1 en una familia previamente caracterizada por análisis de ligamiento. La mutación consiste en una sustitución de una base C por una T en la posición 12220 originado un codón stop donde se produce la mutación. Esto llevaría a una terminación prematura en la traducción produciendo una proteína en la cual estaría ausente parte del extremo carboxílico.

Control Mechanism for Production and Activation of Helicobacter pylori Urease

To define the genes for production of catalytically active H. pylori urease, we camed out study to elucidate the structure of urease gene transcript, to delineate the genetic region which affected the extent of the expression and the activation of urease structural subunits. UreC and ureD were confirmed not to affect the expression of structural genes and active enzyme production, meaning that these genes are not components of the urease gene cluster of H. pylori. p-independent transcriptional stop signal was found in 12 bp down-stream of ureH stop codon. RNA extension test showed that the transcript starts with 267 bp upstream of ureA start codon. Although accessory genes did not affect the extent of the expression of the structural subunits, they were essential for assembling the active urease in E. coli. E. coli transformants of plasmid clones containing ureAB produced catalytically active urease when they are complemented with the plasmid clones of ureIEFGH or coexisted with ureIEFGH, meaning that accessory gene products could be trans-acting as well as cis-acting. The extent of production of urease structural subunits depended on the region of 241 to 57 bp upstream of ureA start codon. E. coli transformant of pBeloBACII clone containing the urease gene cluster, which is maintained with a single copy in host, did not express the urease. Proteins (60, 38, 30, 29, 27, and 24 kDa) that could hold nickel ions were identified in the cell extract of H. pylori. The results in this study will provide the basis to understand the control mechanism for urease gene expression and formation of the active urease.

Molecular Cloning and Nucleotide Sequence of a Gene Encoding Alcohol Dehydrogenase of Helicobacter pylori

Partially purified H. pylori ADH was used to determine the amino acid sequence of ADH N- terminus. The sequence of the ADH N-terminus was determined as MRVQSKGF. The genomic library of H. pylori that has been prepared by pTZ19U plasmid vector was screened with the deduced oligonucleotide probes to select the plasmid clone containing the entire ADH gene. The clone pTZ19U/ADH-6 was selected and its EcoRI-BamHI fragment (1.3 kb) was subcloned into pBluescript II K/S vector to determine nucleotide sequence. The length of H. pylori ADH gene was 1,044 bp. Ribosomal binding site was found in the upstream of start codon and rho- independent transcriptional stop signal was observed in the downstream of stop codon. The ADH gene encodes a protein of 348 amino acids, of which the predicted molecular size and pI value were 38.6 kDa and 7.1, respectively. ADH activity of E. coli transformant of pBluescript/ADH is 10-times greater compared to that of non-transformants. When H. pylori ADH gene was disrupted by pBluescript/ADH-KM whose internal region of 1.3 kb DNA fragment containing ADH gene was replaced by KM resistance sequence, the strain lost the ADH activity completely, despite the normal growth of the strain. This demonstrates that ADH gene is not essential for the viability of H. pylori.