BigDye Terminator Cycle Sequencing Kit Research Articles

Hepatitis B Transmission: Is Vertical Transmission the Major Route in Intermediate Endemic Areas? A Proof-of-Concept Study Based on Mother–Child Genotypes

The route of hepatitis B transmission is believed to be horizontal in India, though pediatric studies showed mother as source in the majority of chronic HBV (CHB) cases. We aimed at establishing the fact that mother-child transmission is the main route of acquisition by documenting genotypically identical viruses in mother-child pairs. Blood samples of consecutive children (≤ 18 years) with CHB and high DNA (> 10,000 IU/mL) and their positive mothers were collected from January 2013 to December 2015. Polymerase chain reaction (PCR) products of HBV-DNA were amplified and sequenced by using BigDye Terminator Cycle Sequencing Kit v3.1 and aligned with previously described sequences in the region of interest for genotypes A to G by using BioEdit software. Phylogenetic tree was generated using p-distance algorithm in MEGA software version 6. Genotyping of 59 (33 children and 26 mothers) subjects include genotype A in 24 (40.7%) and genotype D in 35 (59.3%). Both mother-child pair genotyping was possible in 25. The median age of 25 children (20 males) was 9 (interquartile range, IQR: 4-11) years. The distribution of genotypes among mother-child pairs was similar. The concordance between children and their mothers was 24 of 25 (96%). Evolutionary analysis showed significant similarities between mother and child sequences for both genotype A and D, suggesting thereby the same virus. In conclusion, mother-baby transmission seems to be the major route of acquisition of HBV in children in India and near-complete homology in genetic sequences between mother-child pairs is definite proof for that. However, a larger epidemiological study is required to substantiate our findings.

The influence of high temperature on the possibility of DNA typing in various human tissues.

The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to in­vestigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 100–1000°C for 5 and 10 minutes. Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates.

Biological network inferences for a protection mechanism against familial Creutzfeldt-Jakob disease with E200K pathogenic mutation.

BackgroundHuman prion diseases are caused by abnormal accumulation of misfolded prion protein in the brain tissue. Inherited prion diseases, including familial Creutzfeldt-Jakob disease (fCJD), are associated with mutations of the prion protein gene (PRNP). The glutamate (E)-to-lysine (K) substitution at codon 200 (E200K) in PRNP is the most common pathogenic mutation causing fCJD, but the E200K pathogenic mutation alone is regarded insufficient to cause prion diseases; thus, additional unidentified factors are proposed to explain the penetrance of E200K-dependent fCJD. Here, exome differences and biological network analysis between fCJD patients with E200K and healthy individuals, including a non-CJD individual with E200K, were analysed to gain new insights into possible mechanisms for CJD in individuals carrying E200K.MethodsExome sequencing of the three CJD patients with E200K and 11 of the family of one patient (case1) were performed using the Illumina HiSeq 2000. The exome sequences of 24 Healthy Koreans were used as control. The bioinformatic analysis of the exome sequences was performed using the CLC Genomics Workbench v5.5. Sanger sequencing for variants validation was processed using a BigDye Terminator Cycle Sequencing Kit and an ABI 3730xl automated sequencer. Biological networks were created using Cytoscape (v2.8.3 and v3.0.2) and Pathway Studio 9.0 software.ResultsNineteen sites were only observed in healthy individuals. Four proteins (NRXN2, KLKB1, KARS, and LAMA3) that harbour rarely observed single-nucleotide variants showed biological interactions that are associated with prion diseases and/or prion protein in our biological network analysis.ConclusionThrough this study, we confirmed that individuals can have a CJD-free life, even if they carry a pathogenic E200K mutation. Our research provides a possible mechanism that involves a candidate protective factor; this could be exploited to prevent fCJD onset in individuals carrying E200K.

Evaluation of anthropogenic effects on water quality and bacterial diversity in Rawal Lake, Islamabad

Water quality and bacterial diversity in the surface water of Rawal Lake was investigated for a period of 8 months to evaluate the pollution load from anthropogenic effects of surrounding areas. Rawal Lake in Islamabad, Pakistan is an artificial reservoir that provides the water needs for the residents of Rawalpindi and Islamabad. Grabbed water samples were collected according to standard protocols from ten different locations of the lake and tributaries keeping in view the recharge points from adjacent areas. Temperature, pH, electrical conductivity, dissolved oxygen, total dissolved solids, hardness, alkalinity, and turbidity of water samples were determined to study the water quality characteristics. The physicochemical parameters showed higher values at the tributaries as compared to the sampling locations within the lake such as values of hardness and alkalinity were 298 and 244 mg/L, respectively, at the tributary of the Nurpur stream. Bacterial strains were isolated by streaking on differential and selective growth media by observing colony morphology and other biochemical tests such as Gram reaction, oxidase, and catalase test. Template DNA was prepared from pure cultivated bacteria and 16S rRNA gene analysis was performed using universal primers for bacteria. Sequencing was performed by using BigDye terminator cycle sequencing kit. Sequences of nearest relative microbial species were identified by using basic local alignment search tool and used as reference sequences for phylogenetic analysis. Phylogenetic trees were inferred using the neighbor-joining method. Sequencing and phylogenetic characterization of microbes showed various phylotypes, of which Firmicutes, Teobacteria, and Proteobacteria were predominant.

Clinical and Molecular Profile Of Hereditary Antithrombin (AT) Deficiency: A Single Institution Cohort

IntroductionHereditary antithrombin (AT) deficiency is an autosomal dominant thrombophilia. It is classified into type 1 (quantitative) or type 2 (qualitative) deficiency based on the AT antigen and activity levels (Haemophilia 2008:14; 1229). The goal of this study was to correlate clinical phenotype with AT molecular defects. MethodsAfter IRB approval, patients with a diagnosis of hereditary AT deficiency established at the Mayo Clinic, Rochester, were identified from the clinical database (1997-2012). AT activity was assayed by a chromogenic Factor Xa assay and AT antigen level was assayed by latex immunoassay. Peripheral blood leukocyte genomic DNA was extracted using standard methods.PCR-amplification and ABI BigDye Terminator cycle sequencing kit was performed for all SERPINC1 exons, intronic splicing regions and the 3'UTR. SERPINC1 sequence was analyzed using Mutation Surveyor software (SoftGenetics). Clinical data were obtained from patient interview and medical record review. An event was defined as an episode of venous thromboembolism (VTE), including deep vein thrombosis and/or pulmonary embolism; arterial thrombosis, including cerebrovascular event and/or myocardial infarction, and an obstetric event, including miscarriages and/or spontaneous abortions. Statistical analysis was performed with SAS 9.1.3 (SAS Institute Inc. Cary, NC). ResultsOf 30 patients with hereditary AT deficiency; sequence data was available on 22. Twenty-nine (97%) patients were white, and 19 (63%) were females. Six patients (20%) were smokers and 9 (30%) had a body mass index of >30. Based on the AT activity and antigen levels, 18 (81%) had type 1 AT deficiency, while the remainder had type 2. Eleven patients were heterozygous for six novel (all type 1) mutations. One patient was also a homozygous carrier for the Factor V Leiden mutation.The median age at first thrombotic event was 24.4 years (range, 16.2-70.7), and the median age at diagnosis of AT deficiency was 40.7 years (range, 17.8-76). Both, the median ages at first thrombotic event and at diagnosis were comparable in type 1 versus type 2 AT deficiency patients (P=0.52 and 0.97 respectively). Thirteen patients (43%) had unprovoked thrombotic events. Majority had VTE (n=21, 70%), which included one patient each with splanchnic venous thrombosis, and cerebral venous sinus thrombosis. At last follow up, twenty-one patients (70%) were on chronic anticoagulation [17 (81%) were on warfarin, 2 (9.5%) on enoxaparin and 2 (9.5%) were on rivaroxaban]. Median number of events was 1 (range 0-7). Four patients (19%) had bleeding complications from anticoagulation. Only one death was noted in the cohort and cause of death could not be determined. ConclusionsType 1 hereditary AT deficiency is the most clinically prevalent subtype in practice. Of the patients who developed thrombosis; clinical characteristics did not differ between type 1 and type 2 AT deficiency. We report 6 novel mutations in patients with hereditary AT deficiency. Disclosures:No relevant conflicts of interest to declare.

Genetic Events in Ikaros Gene Family (IKZF1 and IKZF2) in Pediatric T-Cell Lymphoblastic Leukemia/Lymphoma

Abstract 4622The Ikaros gene family, which includes Ikaros (IKZF1), Helios (IKZF2), and Aiolos (IKZF3), is a primary regulator of lymphocyte differentiation and is involved in the development of acute lymphoblastic leukemia (ALL). In particular, loss of IKZF1 is primarily associated with T-cell ALL (T-ALL) development in mouse models. Moreover, Helios expression has been detected in early T-cell lineage and is involved in human lymphoid proliferative disease. We sought to determine whether the Ikaros gene family is associated with leukemogenesis in T-ALL and T-cell non-Hodgkin lymphoma (T-NHL) in Japanese children.Fifty-eight Japanese children (39 boys and 19 girls; age, <18 years) with T-ALL (n = 50) and T-NHL (n = 8) were evaluated. The median age at diagnosis was 8 years (1–15 years). Samples were collected at Japanese Nagoya First Red Cross Hospital and Nagoya University Hospital. Blasts from bone marrow (n = 40), peripheral blood (n = 9), pleural effusions (n = 6), and lymph nodes (n=3) were subjected to DNA extraction. All coding exons in IKZF1 were amplified by polymerase chain reaction using the genomic DNA of patients with T-ALL/T-NHL. After amplification using a BigDye Terminator Cycle Sequencing kit (Life Technologies, Carlsbad, CA, USA) in the second step, products were analyzed in an ABI/PRISM 3130xl Genetic Analyzer (Life Technologies). Multiplex ligation-dependent probe amplification (MLPA) analyses were performed using a SALSA MLPA reaction mixture according to the manufacturer’s protocol [P202 kit; IKZF1(IKAROS); MRC-Holland, Amsterdam, Netherlands]. Amplification products were quantified and identified by capillary electrophoresis on an ABI/PRISM 3130xl Genetic Analyzer. Informed consent was received from parents of the patients. Approval for this study, including molecular analyses, was obtained from the institutional ethics committee of Nagoya University Graduate School of Medicine.An IKZF1 mutation was found in only 1 of 59 (2%) patients (exon 5, c.476 A>G, p.N159S; patient number 41) who was diagnosed with primary immune deficiency after birth. She received regular immunoglobulin replacement therapy just before the development of T-ALL at 13 years of age. Her IKZF1 mutation was confirmed as a germline mutation by sequencing of genomic DNA from her fingernails. In the IKZF1 null mouse model, Ikaros inactivation is closely linked to T-ALL development, which is highly associated with acquisition of a somatic NOTCH1 mutation. As in the mouse model, we could identify a somatic NOTCH1 mutation (exon 26, c.4750, insGAG) in the sample upon T-ALL development. IKZF1 mutations were not found in the other 57 samples. This finding implied that the IKZF1 mutation was a rare genetic event in de novo T-ALL/T-NHL in children. Furthermore, we analyzed all samples by MLPA to determine if monoallelic genomic deletions were present. Although 59 samples exhibited normal biallelic IKZF1 and IKZF3 chromosomal regions, we identified IKZF2 microdeletions in 2 (3.4%) patients (numbers 9 and 49). One of them with an IKZF2 microdeletion died of leukemia relapse after allogeneic bone marrow transplantation from a human leukocyte antigen (HLA)-matched sibling donor. Overexpression of full-length Helios (IKZF2) blocks αβ T-cell differentiation at the CD4-CD8- stage in the thymus, and results in the increased frequencies of γδ T and natural killer (NK) cells in peripheral lymphoid organs. Overexpression of dominant negative Helios leads to increased T-cell proliferation upon T-cell receptor stimulation and the development of T lymphoma. Furthermore, dominant negative Helios isoforms or allelic losses have been detected in some human T-ALL/T-NHL. These results suggest that Helios is an essential regulator of T-cell homeostasis and a tumor suppressor.In general, IKZF1 mutation or deletion is a rare event in pediatric patients with T-ALL/T-NHL. We could identify a secondary T-ALL patient after primary immunodeficiency who shared genetic findings with an IKZF1 null mouse model (germline IKZF1 mutation and somatic NOTCH1 mutation). We found IKZF2 microdeletion in 2 of 59 patients. The genetic events in the Ikaros gene family (IKZF1 and IKZF2) are associated with leukemogenesis in a few pediatric patients T-ALL/T-NHL. Disclosures:No relevant conflicts of interest to declare.

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

Glycogen storage diseases are a group of inborn errors of glycogen synthesis or catabolism. The outcome for untreated patients can be devastating. Given the genetic heterogeneity and the limited availability of enzyme study data, the definitive diagnosis of glycogen storage diseases is made on the basis of sequence analysis of selected potentially causative genes. A massively parallel sequencing test was developed for simultaneous sequencing of 16 genes known to cause muscle and liver forms of glycogen storage diseases: GYS2, GYS1, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, and PGM1. All the nucleotides in the coding regions of these 16 genes have been enriched with sufficient coverage in an unbiased manner. Massively parallel sequencing demonstrated 100% sensitivity and specificity as compared with Sanger sequencing. Massively parallel sequencing correctly identified all types of mutations, including single-nucleotide substitutions, small deletions and duplications, and large deletions involving one or more exons. In addition, we have confirmed the molecular diagnosis in 11 of 17 patients in whom glycogen storage diseases were suspected. This report demonstrates the clinical utility of massively parallel sequencing technology in the diagnostic testing of a group of clinically and genetically heterogeneous disorders such as glycogen storage diseases, in a cost- and time-efficient manner.

Prothrombin Mumbai causes severe prothrombin deficiency due to a novel Cys90Ser mutation

Dear Editor, Prothrombin (coagulation factor II) is a zymogen which is proteolytically cleaved to form its active derivative, thrombin, a serine protease, in the penultimate step of the coagulation cascade. The prothrombin gene is located on chromosome 11 (11p11-q12) [1]. Homozygous prothrombin deficiency is a rare autosomal recessive bleeding disorder occurring approximately in one of two million individuals. The Human Genome Mutation Database shows reports of only 32 mutations causing prothrombin deficiency [2]. The molecular basis is diverse, and mutations or polymorphisms in f2 gene are known to lead to both thrombosis and bleeding [3, 4]. A 5-month-old female child from Nepal, born of nonconsanguinous marriage, was referred with acute intracranial hemorrhage (Fig. 1a). Since birth, the baby had few episodes of bleeding ranging from hematemesis, umbilical stump bleed, and right-sided subdural hematoma. During these episodes, the baby was given an injection of vitamin K, fresh frozen plasma, and whole blood. Coagulation investigations done during these episodes showed consistently deranged prothrombin time (PT) and activated partial thromboplastin time (APTT); however, no reports of factor assays were available. Both the parents were clinically normal with neither any history of bleeding nor thrombosis during their life time. Blood samples of the patient and parents were collected in 3.2 % tri-sodium citrate after an informed consent. All the coagulation tests were performed in a semi-automated coagulometer using commercial reagents and calibrators (Diagnostica Stago, Asnieres, France). All the 14 exons and their flanking intronic regions were amplified using 12 sets of primers as described earlier [5]. The amplicons were sequenced using BigDye Terminator cycle sequencing kit in a ABI 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). The primary amino acid sequences of prothrombin from Homo sapiens (NC_000011.9), Pan troglodytes (NC_006478.2), Macaca mulatta (NC_007871.1), and Mus musculus (NC_000068.6) were obtained from NCBI gene [6]. Multiple sequence alignment was performed with ClustalW [7] to study the conservation of amino acids mutated by missense variations. Ensembl Genome Browser [8] was used to refer to the reported variations in the prothrombin gene. PolyPhen, Sorting Intolerant from Tolerant (SIFT), and Panther were used to predict the severity of the amino acid substitution on the protein function. The coagulation parameters of the patient were as follows: PT 27 s/control 14.5 s, APTT 40 s/control 29 s, thrombin time (TT) 18 s/control 15 s, fibrinogen 222 g/dL (normal range, 150–400 mg/dL). Mixing studies were normal for PT, APTT, and TT. Factor II (prothrombin) was found to be <1% on assay, with other factors being within normal limits. PCR amplification of all 14 exons followed by DNA sequencing in the patient showed a homozygous c.G269C variation in exon 4 of the patient. Both the parents were B. Kulkarni :K. Ghosh : S. Shetty (*) National Institute of Immunohaematology (I.C.M.R.), 13th floor, New building, K.E.M. Hospital Campus, Parel, Mumbai 400012, India e-mail: shrimatishetty@yahoo.com

First Report of Calonectria Leaf Spot Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Rhododendron in Belgium.

Belgium is one of the most important Rhododendron-producing areas in Europe, with an annual sale of approximately 1.6 million plants. In June 2010, an outbreak of leaf spots on several thousands of Rhododendron cv. Marcel Menard plants took place at a nursery near Gent. Diseased plants showed dark brown leaf spots that enlarged and finally resulted in leaf drop. Symptoms developed most explicitly on this cultivar, especially after standard repotting during May or June and when repotting was followed by a few days of unusually warm temperatures (30 to 35°C). The leading edge of diseased leaf tissue was excised, surface disinfected with 1% NaOCl for 60 s, and rinsed twice with sterile distilled water before being plated onto potato dextrose agar (PDA). After 5 days of incubation at 21°C in the dark, Cylindrocladium-like fungal colonies with white aerial mycelium and amber-brown growth within the agar consistently developed. Mycelium was transferred aseptically to fresh plates of PDA and incubated for 10 to 14 days at 17°C under a 12-h fluorescent light regimen to study the morphological characteristics. Conidiophores showed a penicillate arrangement of fertile branches, producing two to six phialides. They arose from a stipe and terminated in a clavate vesicle (3 to 5 μm). Conidia were straight, cylindrical, rounded at both ends, three septate, and measured 60 to 70 × 4 to 6 μm. Yellow subglobose to oval perithecia were abundantly produced. Asci were clavate, four spored, and measured 100 to 150 × 15 to 30 μm. Ascospores were hyaline, three septate, and measured 50 to 65 × 5 to 6 μm. These characteristics are consistent with those of Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii) (1). The β-tubulin gene was PCR-amplified with DNA extracted from the mycelium and the T1 and T2 primers (3), sequenced directly with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA), and the DNA sequence was deposited (GenBank Accession No. JF802784). BLASTn alignment showed 99% identity (525 of 526 nucleotides) with the β-tubulin DNA sequence derived from Calonectria colhounii CBS 293.79 (GenBank Accession No. DQ190564). A spore suspension (105 conidia per ml) was prepared from a 1-week-old culture, and 50-μl drops were used to inoculate the abaxial side of 10 detached 1-year-old leaves from Rhododendron cv. Cunningham's White. Ten control leaves were inoculated with water. The leaves were placed in a moist chamber and incubated at 21°C in the dark. After 5 to 6 days, all spore-inoculated leaves showed lesions identical to those on the naturally infected leaves, while the water-inoculated leaves remained symptom free. Following the original procedure, the fungus was reisolated from the diseased leaves and the morphological characteristics of the resulting culture were the same as those of the inoculated isolate, completing Koch's postulates. This fungus has been described on Rhododendron in the United States (2), but to our knowledge, this is the first record of Calonectria colhounii on Rhododendron in Belgium.

Saffold Cardioviruses in Children with Diarrhea, Thailand

To the Editor: Cardioviruses currently consist of at least 3 viruses: Theiler murine encephalomyocarditis virus, encephalomyocarditis virus, and Saffold virus (SAFV) (1–4). Saffold cardiovirus in the family Picornaviridae was isolated and identified from fecal specimens of a child with fever of unknown origin in the United States (3). Several reports have documented the presence of SAFV in fecal samples and respiratory secretions (5–10). However, it is not clear whether SAFV is associated with any disease, including gastroenteritis in humans, and epidemiologic data for SAFV are limited. We report an epidemiologic survey of SAFV in children hospitalized with diarrhea in Chiang Mai, Thailand. A total of 150 fecal specimens were obtained from children hospitalized with acute gastroenteritis in Chiang Mai during January–December 2007. Patient ages ranged from >1 to 5 years. SAFV in fecal specimens was detected by using a nested PCR and primers specific for the virus 5′ untranslated region (7). A negative control was also included to monitor any contamination that might have occurred during the PCR. SAFVs detected were further analyzed by amplification of the viral protein (VP) 1 gene (6,9,10) and direct sequencing of the VP1 PCR amplicon by using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). VP1 sequence was compared with VP1 sequences of reference strains available in the National Center for Biotechnology Information (Bethesda, MD, USA). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA4 (www.megasoftware.net). Nucleotide sequences of SAFV strains described were deposited in GenBank under accession nos. {type:entrez-nucleotide-range,attrs:{text:HQ668170-HQ668173,start_term:HQ668170,end_term:HQ668173,start_term_id:335347130,end_term_id:335347136}}HQ668170-HQ668173. Four (2.7%) of 150 specimens were positive for SAFV (CMH023/2007, CMH038/2007, CMH045/2007, and CMH143/2007). Two of these specimens (CMH023/2007 and CMH038/2007) were obtained in February 2007, one (CMH045/2007) in March 2007, and 1 (CMH143/2007) in November 2007. Co-infections with other viruses were detected in all 4 samples. Two specimens (CMH023/2007and CMH045/2007), were co-infected with noroviruses GII/16 and GII/4 genotypes, respectively. One SAFV-positive sample (CMH038/2007) was co-infected with a group A rotavirus G1P[8] genotype, and another (CMH143/2007) was co-infected with human parechovirus. All SAFV-positive specimens were further amplified for the VP1 gene to determine their phylogenetic lineages and genetic relationships with other SAFV reference strains. When we used 3 sets of primers used in other studies (6,9,10) for amplification of the VP1 gene, this gene was amplified only by the primer set reported by Itagaki et al. (10). Analysis of partial VP1 sequences (369 nt) of 4 SAFV strains showed that strains CMH023/2007 and CMH143/2007 were highly conserved (nt sequence identities >97%). These 2 SAFV strains were most closely related to the prototype strain of SAFV1 ({type:entrez-nucleotide,attrs:{text:EF165067,term_id:172050184,term_text:EF165067}}EF165067) isolated in the United States (nt sequence identity range 87.6%–88.9%) and SAFV strains from China (LZ50419, BCH895, GL311, and GL377) (Figure). In addition, the other 2 SAFVs identified in the present study (CMH038/2007 and CMH045/2007) were identical to each other and closely related to SAFV2 strains from China (BCHU79, BCHU353) and Finland (Finland 2008, FIN08–13B) (nt sequence identity range 94.8%–95.6%). Phylogenetic analysis showed that CMH038/2007 and CMH045/2007 were clustered within the SAFV2 lineage (Figure). Figure Phylogenetic analysis of the partial nucleotide sequence (369 nt) encoding the viral protein 1 gene of Saffold virus (SAFV) isolated in this study and other reference strains. The tree was generated by using the neighbor-joining method and MEGA4 (www.megasoftware.net ... The 4 strains of SAFV were isolated from children with acute gastroenteritis who were co-infected with other viral pathogens (norovirus, group A rotavirus, and human parechovirus). Therefore, we could not determine whether SAFVs identified in this study were associated with acute gastroenteritis. The detection rate for SAFV in children with acute gastroenteritis (2.7%) in our study was consistent with that in a study in Beijing, People’s Republic of China (3.2%) (9). Phylogenetic analysis of the VP1 region demonstrated that 2 SAFV lineages (SAFV1 and SAFV2) were circulating in Chiang Mai, Thailand. Further extensive epidemiologic surveillance of SAFV in other areas may provide a better understanding of the distribution, heterogeneity, and association of SAFV with enteric diseases in humans.

RAD51C germline mutations in Chinese women with familial breast cancer

Germline mutations in high penetrance breast cancer susceptibility genes, BRCA1 and BRCA2, account for only approximately 10% of Chinese familial breast cancers [1], indicating other susceptibility genes related to Chinese familial breast cancer may exist. RAD51C gene (Rasrelated associated with diabetes) is located on chromosome 17q23, a region that was found to be frequently amplified in breast tumors [2, 3]. RAD51C gene is a member of the RAD51 family and plays an essential role in homologous recombination, DNA damage sensitivity, genomic integrity, embryonic development, activation of cell cycle checkpoint kinase 2 (CHK2), and cell cycle arrest in response to DNA damage [4–8]. Recently, a biallelic missense mutation in the RAD51C gene was found in a family from Pakistan with Fanconi anemia-like disorder [9]. In an accompanying article, six monoallelic deleterious mutations in the RAD51C gene were found in 480 BRCA1/2-negative breast/ovarian cancer families from Germany [10]. This study provides the first evidence to show that the RAD51C is a breast cancer susceptibility gene. Thus, the RAD51C gene may deserve to be comprehensively screened as a breast cancer susceptibility gene in other populations. In this study, we screened the entire coding regions and exon–intron boundaries of the RAD51C gene in 273 Chinese women with familial breast cancer who do not carry mutations in BRCA1 and BRCA2 genes by polymerase chain reaction (PCR)-sequencing. Familial breast cancer cases were from the Breast Center, Peking University Cancer Hospital from July 2006 to May 2010. The criteria of familial breast cancer are (1) patients have at least one or more firstor second-degree relatives affected with breast cancer and/or ovarian cancer regardless of age and (2) bilateral breast cancer regardless of age. Four hundreds seventy-five healthy Chinese women serve as controls. Both of the cases and controls are Han Chinese women and reside in the north of China. The whole coding sequences of RAD51C were amplified using nine sets of primers described elsewhere with a minor modification [10]. All fragments were sequenced using BigDye Terminator Cycle Sequencing Kit and ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA). Each mutation was confirmed by duplicate. In total, we detected eight germline sequence variants in the RAD51C gene in the 273 BRCA1/2-negative familial breast cancer cases. Among them, three were non-coding variants and five were coding variants (Table 1). None of them were previously reported. Of the coding variants, one was synomenous and four were non-synomenous. Among the four amino-acid substitution variants, 4C[G (R2G) was located in exon 1, 635G[A (R212H) and 644A[G (D215G) in exon 4, and 882G[C (Q294H) in exon 6. The missense variants R2G, D215G, and Q294H were detected in one index case, whereas R212H was detected in two unrelated cases. We then screened the four missense variants in 475 healthy controls. The variant R212H was found in two healthy individuals, indicating this variant was unlikely to be pathogenic. In contrast, none of the Zhiyuan Pang and Lu Yao contributed equally to this study.

Rare Rotavirus Strains in Children with Severe Diarrhea, Malaysia

Rare Rotavirus Strains in Children with Severe Diarrhea, Malaysia

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Abstract P3-14-11: Trastuzumab Cardiac Toxicity and HER2 Polymorphism: A Case-Control Study

Abstract Background: Trastuzumab (T) is a very effective targeted therapy used in breast cancer patients but it can induce cardiac toxicity. Gene polymorphisms of Her 2 have been identified. According to a small report of Beauclair (2007), the polymorphism at codon 655 of the HER2 gene may represent a risk factor for cardiotoxicity induced by T. In this trial, we studied the incidence of the Val655Ile polymorphism in breast cancer patients with cardiac toxicity and in matched contol patients without cardiac toxicity. Patients and methods: In our institution, between January 1st, 2007 and April 15th, 2010, 26 patients developed cardiac toxicity potentially related to T and were referred to a cardiologist and treated with angiotensinconverting enzyme inhibitors, Beta-blockers. Their cardiac function was monitored. Twenty two control pts without cardiac toxicity were matched according to age, received dose of anthracyclin, BMI, cardiac risk factors, cardiac treatment and initial left ventricular ejection fraction. Her2 gene polymorphism in codon 655 was studied by PCR on lymphocyte DNA from blood samples in both populations. Genomic DNA was extracted from EDTA blood samples (300μl) on the MagNa Pure Compact instrument (Roche Diagnostics, Meylan, France), using the MagNA Pure Compact Nucleic Acid Isolation Kit according to the manufacturer's instructions. The HER2 Ile655Val (rs1163201) polymorphism was detected by sequencing using a 3730 DNA Analyzer (Applied Biosystems, Courtaboeuf, France) with the BigDye Terminator Cycle Sequencing kit and universal primers M13. Results: The mean age was 54.5 (31-72) and 52.5 (37-73) years respectively in the cardiac toxicity group and in the control group. In the cardiac toxicity group, 17 patients were in an adjuvant or a neoadjuvant setting, 9 were treated for a metastatic disease; 7 had cardiac risk factors (hypertension, diabetes, dyslipidemia or cardiac familial event). The control pts had similar characteristics. In the cardiac toxicity group 3.9% (1 patient) presented a genotype Val/Val at Her 2 codon 655, 42.3% (11)were heterozygous Ile and 53.8% (14) had the wild type. In the control population, the incidence was respectively 4.5% (1 patient), 31.8% (7) and 63.7% (14). No differences were found between the both groups. Discussion-Conclusion: The incidence of mutated, heterogeneous and wild type genotype was similar as the in the literature. We could not confirm the cardiac risk related to the Val allele at Her2 codon 655. Her2 polymorphism cannot be used in clinical practice to predict the risk of cardiac toxicity of Trastuzumab. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-11.

Structure-based Catalytic Optimization of a Type III Rubisco from a Hyperthermophile

The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high temperatures. We have previously found that replacement of the entire α-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic temperatures, both in vivo and in vitro, whereas the former and latter half-replacements of the α-helix 6 (SP4 and SP5 mutants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of α-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the α-helix 6 and loop 6 regions was important to increase the catalytic activity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5-V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures.

Novel fibrinogen mutation γ314Thr→Pro (fibrinogen AI duPont) associated with hepatic fibrinogen storage disease and hypofibrinogenaemia

Mutation in fibrinogen genes may lead to quantitative or qualitative disorders that result in bleeding, thrombosis or hepatic fibrinogen storage disease. Only three mutations in the fibrinogen γ gene have been identified that cause hepatic endoplasmic reticulum storage of mutant fibrinogen. To investigate the possibility of hepatic fibrinogen storage disease in a 4-year-old male with persistently elevated serum aminotransferases and preserved synthetic function except for a prolonged INR. After informed consent, liver and blood samples were obtained. Liver sections were examined by light microscopy, anti-fibrinogen immunolabelling and electron microscopy. Purified fibrinogen was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography; DNA sequencing was performed using a BigDye Terminator (v. 3.1) cycle sequencing kit. Four-year-old male with persistently elevated transaminases with an INR 1.5 but otherwise normal synthetic function. Fibrinogen activity and thrombin clotting time were abnormal at 0.47 g/L and 46 s respectively. Hepatic histological examination revealed portal inflammatory infiltrates with bridging fibrosis. Clumped eosinophilic material was observed in hepatocytes that was immunoreactive to fibrinogen antisera. Ultrastructural examination showed cytoplasmic inclusions arrayed in fingerprint-like patterns. DNA sequence analysis revealed heterozygosity for a novel γ314Thr →Pro mutation (fibrinogen AI duPont) in the fibrinogen γ gene. Protein analyses showed normal patterns of Aα, Bβ and γ chains suggesting that the variant γ allele was not expressed in plasma fibrinogen. We describe only the fourth mutation to be identified, γ314Thr→Pro (fibrinogen AI duPont), giving rise to hypofibrinogenaemia and hepatic fibrinogen storage disease.

Fluoroquinolone-Resistant Typhoid, South Africa

Fluoroquinolone-Resistant Typhoid, South Africa

A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss

More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss.

FactorVIII Gene Modifications Observed in Acquired Hemophilia.

Abstract 4450Acquired hemophilia is a rare bleeding disorder caused by autoantibodies directed against the coagulation factor VIII (FVIII). Usually acquired hemophilia is found in the elderly person, autoimmune disorder or women in the postpartum. Incontrast to alloantibody inhibitors of hereditary hemophilia, acquired inhibitor is presumably due to a spontaneous autoimmune disorder in which patients with previously normal hemostasis develop autoantibodies against FVIII. However, the precise cause of the antibody formation remains obscure and the possibility of abnormal FVIII protein derived from the FVIII gene mutation might have caused the antibody formation was investigated by direct FVIII gene sequencing method. Blood samples were obtained from a 72 year old male who initially presented with a large hematoma in the Rt. Shoulder, and had a prolonged aPTT (46 sec), FVIII level of 14%, and VIII inhibitor titer 2∼5 BU/ml. Direct sequencing of FVIII was performed via amplification for all 26 exons, including the 5′-UTR and 3′-UTR region, using the 35 synthesized primers. Sequencing was conducted using BigDye Terminator cycle sequencing kit on an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA). Sequence variations were analyzed with CLC main workbench program (CLC, Aarhus. Denmark). Firstly, we analyzed translational region of FVIII gene. But we couldn't find any sequence variation. Then we analyzed the sequence of untranslationed region, 5′-UTR and 3′-UTR. As a result, we identified gene variation of substitution to c.8889G>A in the 3′-UTR. We speculate that this variation may have affected the RNA processing or mRNA stability. Thus we need to study about the relationship between this sequence variation and FVIII function. Also, we will investigate whether this gene variation is functional mutation. The cause of acquired hemophilia remains obscure. Variations in FVIII molecules including in untranslatable 5′- or 3′-region should be studied further to delineate the cause of antibodies in acquired HA. We expect that the accumulation of sequence variation can help to understand the cause of acquired hemophilia through this study. Disclosures:No relevant conflicts of interest to declare.

First Report of Soybean yellow mottle mosaic virus in Soybean in North America.

Soybean yellow mottle mosaic virus (SYMMV) is a soybean-infecting virus recently discovered in Korea that initially induces bright yellow mosaic on leaves followed by stunting and reduced growth of older leaves (1). Nucleotide sequence analysis of genomic RNA of the Korean SYMMV isolate suggested that the virus is a new member of the genus Carmovirus in the family Tombusviridae. To determine whether SYMMV is present in the United States, single leaflets were collected without regard for symptoms from 7 to 10 plants in each of 136 plots in August 2008 from a research field in Stoneville, MS that contained 16 plant introductions (including five from Korea) and 'Williams 82'. Samples were grouped into 10 pools of 100 leaves from which total RNA was extracted with the Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScript III Platinum SYBR Green One-Step Quantitative Real-time Reverse Transcriptase-PCR Kit (Invitrogen, Carlsbad, CA) and two pairs of oligonucleotide primers (5'-CGTCTGCCAGGGTTTAATACTA-3', and 5'-GATTAGCATGTCAGGGTGGTCG-3'; and 5'-ACTGAGTCCCCTGCTTAT-3' and 5'-CATCACTAGCGTCYGGATCA-3') that were designed from regions conserved between SYMMV and Cowpea mottle virus (CPMoV; a related and seed-transmitted carmovirus). Six 100-leaflet pools were positive with both primer sets and four pools were negative with both primer sets. Total RNA extracted from one positive pool was reverse transcribed using SuperScript II reverse transcriptase and a primer complementary to nt 4,000 to 4,009 of the SYMMV genome and amplified using iProof DNA polymerase (Bio-Rad, Hercules, CA) as two overlapping DNA fragments using primers corresponding to nt 1 to 21 and complementary to nt 3,483 to 3,508 and corresponding to nt 3,366 to 3,391 and complementary to nt 4,000 to 4,009. DNA fragments were sequenced using a BigDye Terminator Cycle Sequencing Kit and ABI 3730XL capillary sequencers (Applied Biosystems, Foster City, CA). The 4,009-nt sequence of the Mississippi SYMMV isolate (GenBank Accession No. FJ707484) was 96% identical to the Korean SYMMV isolate and 65% identical to CPMoV. Because of the sampling techniques used, it was not possible to associate SYMMV-positive plants with disease symptoms in Mississippi. To our knowledge, this is the first report of SYMMV in North America. Reference: (1) M. Nam et al. Online publication. doi:10.1077/s00705-009-0480. Arch. Virol., 2009.