Number Tandem Repeat Research Articles

Country-wide genotyping of Mycobacterium tuberculosis complex in Singapore, 2011–2017

ObjectiveTo describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore. MethodsSpoligotyping, 24-loci mycobacterial interspersed repetitive units – variable number of tandem repeats (MIRU-VNTR) typing and demographic data from the national TB notification registry of MTBC culture-positive cases notified from January 2011 to December 2017 were analysed. ResultsOf the 12,046 culture-positive cases notified, complete spoligotyping and MIRU-VNTR typing results were available for 8690 (72.1%) belonging to 4950 (57.0%) local-born and 3740 (43.0%) foreign-born persons. From these, 4810 (55.3%) were identified in 883 clusters. The proportion of recent transmission was 45.2%. The East-Asian Lineage 2 accounted for 4045 (47.1%) of isolates, and disproportionately accounted for large clusters. Clustered cases were more likely to be older than 50 years, male, Malay, local-born, Singapore citizens or Permanent Residents, of lower socioeconomic status, imprisoned; to harbour East-Asian Lineage 2 strain; to have cavitary pulmonary TB, positive sputum smear or be recalcitrant treatment defaulters. They were less likely to have multidrug-resistant, or isoniazid or rifampicin mono-resistant TB. ConclusionWe demonstrated the diversity of MTBC strains and, notwithstanding the likely over-estimation of clustering using these genotyping methods, elucidated factors associated with TB transmission in Singapore.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

The Plastome Sequences of Triticum sphaerococcum (ABD) and Triticum turgidum subsp. durum (AB) Exhibit Evolutionary Changes, Structural Characterization, Comparative Analysis, Phylogenomics and Time Divergence

The mechanism and course of Triticum plastome evolution is currently unknown; thus, it remains unclear how Triticum plastomes evolved during recent polyploidization. Here, we report the complete plastomes of two polyploid wheat species, Triticum sphaerococcum (AABBDD) and Triticum turgidum subsp. durum (AABB), and compare them with 19 available and complete Triticum plastomes to create the first map of genomic structural variation. Both T. sphaerococcum and T. turgidum subsp. durum plastomes were found to have a quadripartite structure, with plastome lengths of 134,531 bp and 134,015 bp, respectively. Furthermore, diploid (AA), tetraploid (AB, AG) and hexaploid (ABD, AGAm) Triticum species plastomes displayed a conserved gene content and commonly harbored an identical set of annotated unique genes. Overall, there was a positive correlation between the number of repeats and plastome size. In all plastomes, the number of tandem repeats was higher than the number of palindromic and forward repeats. We constructed a Triticum phylogeny based on the complete plastomes and 42 shared genes from 71 plastomes. We estimated the divergence of Hordeum vulgare from wheat around 11.04–11.9 million years ago (mya) using a well-resolved plastome tree. Similarly, Sitopsis species diverged 2.8–2.9 mya before Triticum urartu (AA) and Triticum monococcum (AA). Aegilops speltoides was shown to be the maternal donor of polyploid wheat genomes and diverged ~0.2–0.9 mya. The phylogeny and divergence time estimates presented here can act as a reference framework for future studies of Triticum evolution.

Short Tandem Repeat Variation in the CNR1 Gene Associated With Analgesic Requirements of Opioids in Postoperative Pain Management.

Short tandem repeats (STRs) and variable number of tandem repeats (VNTRs) that have been identified at approximately 0.7 and 0.5 million loci in the human genome, respectively, are highly multi-allelic variations rather than single-nucleotide polymorphisms. The number of repeats of more than a few thousand STRs was associated with the expression of nearby genes, indicating that STRs are influential genetic variations in human traits. Analgesics act on the central nervous system via their intrinsic receptors to produce analgesic effects. In the present study, we focused on STRs and VNTRs in the CNR1, GRIN2A, PENK, and PDYN genes and analyzed two peripheral pain sensation-related traits and seven analgesia-related traits in postoperative pain management. A total of 192 volunteers who underwent the peripheral pain sensation tests and 139 and 252 patients who underwent open abdominal and orthognathic cosmetic surgeries, respectively, were included in the study. None of the four STRs or VNTRs were associated with peripheral pain sensation. Short tandem repeats in the CNR1, GRIN2A, and PENK genes were associated with the frequency of fentanyl use, fentanyl dose, and visual analog scale pain scores 3 h after orthognathic cosmetic surgery (Spearman’s rank correlation coefficient ρ = 0.199, p = 0.002, ρ = 0.174, p = 0.006, and ρ = 0.135, p = 0.033, respectively), analgesic dose, including epidural analgesics after open abdominal surgery (ρ = −0.200, p = 0.018), and visual analog scale pain scores 24 h after orthognathic cosmetic surgery (ρ = 0.143, p = 0.023), respectively. The associations between STRs in the CNR1 gene and the frequency of fentanyl use and fentanyl dose after orthognathic cosmetic surgery were confirmed by Holm’s multiple-testing correction. These findings indicate that STRs in the CNR1 gene influence analgesia in the orofacial region.

Genotyping of Listeria monocytogenes isolates by high-resolution melting curve (HRM) analysis of tandem repeat locus

Listeria monocytogenes is responsible for causing listeriosis, a type of food poisoning with high mortality. This bacterium is mainly transmitted to humans through the consumption of contaminated foods. Detection of L. monocytogenes through molecular methods is crucial for food safety and clinical diagnosis. Present techniques are characterized by low discrimination power and high cost, as well as being time-consuming and taking several days to give the final result. In our study, MLVA-HRM (Multiple-Locus Variable-number tandem repeats Analysis ‒ High-Resolution Melting) was investigated as an alternative method for a fast and precise method for the genotyping of L. monocytogenes isolates. Forty-eight isolates of L. monocytogenes obtained from the microbial bank of Department of Microbiology, Iran University of Medical Sciences, were typed by MLVA-HRM analysis using five Variable Numbers of Tandem Repeat (VNTR) loci. A total of 43 different types were obtained. This research demonstrated the usefulness of the MLVA-HRMA method and its ability to discriminate L. monocytogenes isolates. Since this method is easier and more efficient than existing methods, it can be widely used in food processing plants and diagnostic laboratories as a fast and accurate method.

SLC6A3 gene polymorphisms are associated with striatal dopamine transporter changes after glucose loading.

We investigated the association between SLC6A3 gene polymorphisms and changes in dopamine transporter (DAT) availability after glucose loading in humans. An intravenous injection of 18 F-FP-CIT was administered after infusion of glucose or placebo, and the emission data were acquired over 90min in 38 healthy male participants. DAT availability expressed in terms of binding potential (BPND ) was recorded. The 40-bp variable number of tandem repeats (VNTR) in the 3' untranslated region and two single nucleotide polymorphisms (SNPs), rs2652511 and rs2937639, in the SLC6A3 gene were genotyped. Among the 38 participants, those with a VNTR other than 10R/10R (n=7) were excluded. The alleles of the two SNPs (rs2652511 and rs2937639) appeared to be inherited together in two fixed combinations (C-G or T-A) in 29 of 31 individuals. The BPND in the ventral striatum (VST), caudate nucleus, and putamen was not significantly different after glucose or placebo loading according to genotype. However, BPND s from the caudate nucleus and putamen of all participants with rs2652511 CT/rs2937639 AG (n=6) were higher after glucose loading. In conclusion, the SLC6A3 gene polymorphism is associated with the changes in DAT availability after glucose loading. DAT availability after glucose or placebo loading in the VST, caudate nucleus, and putamen did not differ according to the SLC6A3 genotype.

Brucella melitensis biovar 1 isolation in a captive wildlife population in the United Arab Emirates. First isolation in the scimitar-horned Oryx (Oryx dammah)

In 2013, Brucella melitensis biovar 1 was recovered from the stomach contents of a scimitar-horned Oryx - SHO (Oryx dammah) aborted foetus, and from the articular fluid of a sand gazelle (Gazella marica) in a captive wildlife collection near Abu Dhabi, United Arab Emirates. Other evidence of exposure to the pathogen was collected through serological testing (Rose Bengal test) and B. melitensis-specific PCR of samples from captive wildlife kept in six different enclosures. A Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 15 markers showed that the two strains isolated in animals kept in enclosures, located 1300 m apart from each other, shared an identical genotype. The phylogenetic analysis of MLVA-15 profiles retrieved from the public database suggested that these strains belong to the African clade, clustering regionally in the UAE, Oman and Qatar. This is the first confirmed case of B. melitensis in a SHO, an African antelope extinct in the wild and warrants further investigation.

Association of Tandem Repeat Number Variabilities in Subunit S of the Type I Restriction-Modification System with Macrolide Resistance in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is one of the major pathogens responsible for pneumonia in children. Modern molecular genetics has advanced both the management and the epidemiologic study of this disease. Despite these advancements, macrolide resistance remains a global threat in the management of M. pneumoniae infection, for which the genetic background remains unrevealed. In this study, the result of whole genome analysis of 20 sequence type 3 (ST3) M. pneumoniae strains were examined to investigate the gene(s) associated with macrolide resistance. Overall, genetic similarities within M. pneumoniae, and especially ST3, were very high (over 99.99 %). Macrolide resistant ST3 strains shared 20 single nucleotide polymorphisms, of which one gene (mpn085) was found to be associated with resistance. BLAST comparison of M. pneumoniae revealed regular tandem repeat number variabilities between macrolide-susceptible and resistant strains for genes coding the Type I restriction-modification (R-M) system of subunit S (HsdS). Of the ten known HsdS genes, macrolide resistance was determined by the unique tandem repeat of mpn085 and mpn285. In conclusion, the use of whole genome sequencing (WGS) to target macrolide resistance in M. pneumoniae indicates that the determinant of macrolide resistance is variabilities in the tandem repeat numbers of the type I R-M system in subunit S.

Genotyping of Chlamydia abortus using multiple loci variable number of tandem repeats analysis technique

BackgroundThe correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus.MethodsThe aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique.ResultsForty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of aborted fetuses). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6).Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6).ConclusionBy this research revealed that C.abortus was responsible for a significant percentage of ruminant abortion in two studied regions. The main MT type was MT1 (32% of aborted fetuses) and also 7 different genotypes were involved in infections. So it is concluded that diversity in C.abortus genotyping is high in two regions.

Uncovering associations between mental illness diagnosis, nitric oxide synthase gene variation, and peripheral nitric oxide concentration

Nitric oxide (NO) signalling has been implicated in the pathogenesis of several mental illnesses; however, its specific contribution remains unclear. We investigated whether peripheral NO concentration is associated with specific diagnoses, and whether there is a correlation with genetic variation in NO synthase (NOS) genes. We included 185 participants in the study; 52 healthy controls, 43 major depressive disorder (MDD) patients, 41 bipolar disorder (BPD) patients, and 49 schizophrenia (SCZ) patients. Clinical, genetic, and biochemical data were collected at admission to a psychiatric hospital and at discharge. Serum was used to quantify concentration of the stable NO metabolites nitrite and nitrate. Individuals were genotyped for the NOS1 exon 1f variable number of tandem repeats 1 (VNTR1) polymorphism, and single nucleotide polymorphisms (SNPs) in the NOS1, NOS1AP and NOS3 genes. At admission, SCZ patients were found to have significantly higher peripheral NO metabolite (NOx-) concentrations compared to healthy controls, MDD and BPD patients. NOS1 exon 1f VNTR1 short allele carriers were found to have significantly increased NOx- concentration. Moreover, this result was still significant in patients even at discharge. The data also revealed that patients who did not remit in their depressive symptoms had significantly increased NOx- concentration compared to remitters at discharge, supported by the finding of a significant positive correlation between depression symptom severity and NOx- concentration. Taken together, it is possible that elevated peripheral NOx- concentration is associated with increased severity of psychopathology, potentially due to NOS1 exon1f VNTR1 genotype. Our results further implicate NO signalling in mental illness pathogenesis, supporting its possible use as a peripheral biomarker, and imply that NOS genotype may play a significant role in regulating peripheral NOx- concentration.

SITVITBovis—a publicly available database and mapping tool to get an improved overview of animal and human cases caused by Mycobacterium bovis

Limited data are available for bovine tuberculosis and the infections it can cause in humans and other mammals. We therefore constructed a publicly accessible SITVITBovis database that incorporates genotyping and epidemiological data on Mycobacterium bovis. It also includes limited data on Mycobacterium caprae (previously synonymous with the name M. bovis subsp. Caprae) that can infect both animals and humans. SITVITBovis incorporates data on 25,741 isolates corresponding to 60 countries of origin (75 countries of isolation). It reports a total of 1000 spoligotype patterns: 537 spoligotype international types (SITs, containing 25 278 clinical isolates) and 463 orphan patterns, allowing a wide overview of the geographic distribution of various phylogenetical sublineages (BOV_1, BOV_2, BOV_3 and BOV_4-CAPRAE). The SIT identifiers of the SITVITBovis were compared to the SB numbers of the Mbovis.org database to facilitate crosscheck among databases. Note that SITVITBovis also contains limited information on mycobacterial interspersed repetitive units-variable number of tandem repeats when available. Significant differences were observed when comparing age/gender of human isolates as well as various hosts. The database includes information on the regions where a strain was isolated as well as hosts involved, making it possible to see geographic trends. SITVITBovis is publicly accessible at: http://www.pasteur-guadeloupe.fr:8081/SITVIT_Bovis. Finally, a future second version is currently in progress to allow query of associated whole-genome sequencing data.Database URLhttp://www.pasteur-guadeloupe.fr:8081/SITVIT_Bovis

In silico Molecular Characterization of Helicobacter pylori based on Tandem Repeat Number and 16S rRNA gene

In silico Molecular Characterization of Helicobacter pylori based on Tandem Repeat Number and 16S rRNA gene

Laboratory-based study of drug resistance and genotypic profile of multidrug-resistant tuberculosis isolates in Salvador, Bahia, Brazil.

Background: Surveillance of multidrug resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB) is essential to guide disease dissemination control measures. Brazil contributes to a significant fraction of tuberculosis (TB) cases worldwide, but only few reports addressed MDR/XDR-TB in the country. Methods: This cross-sectional, laboratory-based study describes the phenotypic resistance profiles of isolates obtained between January 2008 and December 2011 in Bahia, Brazil, and sociodemographic, epidemiological, and clinical characteristics (obtained from mandatory national registries) of the corresponding 204 MDR/XDR-TB patients. We analyzed the mycobacterial spoligotyping and variable number of tandem repeats of mycobacterial interspersed repetitive units in 12-loci profiles obtained from Salvador. Results: MDR/XDR-TB patients were predominantly male, had a median age of 43 years, belonged to black ethnicity, and failed treatment before MDR-TB diagnosis. Nearly one-third of the isolates had phenotypic resistance (evaluated by mycobacteria growth indicator tube assay) to second-line anti-TB drugs (64/204, 31%), of which 22% cases (14/64) were diagnosed as XDR-TB. Death was a frequent outcome among these individuals and was associated with resistance to second-line anti-TB drugs. Most isolates successfully genotyped belonged to the Latin-American Mediterranean (LAM) Family, with an unprecedented high proportion of LAM10-Cameroon subfamily bacilli. More than half of these isolates were assigned to a unique cluster by the genotyping methods performed. Large clusters of identical genotypes were also observed among LAM SIT42 and SIT376 strains. Conclusions: We highlight the need for strengthening local and national efforts to perform early detection of TB drug resistance and to prevent treatment discontinuation to limit the emergence of drug-resistant strains.

Recognition of Emotional and Neutral Visual Scenes in Carriers of the MAOA, COMT, DRD4, and 5HT2A Gene Polymorphisms

It is known that some genes regulate neurochemical metabolism, and their polymorphisms affect cognitive performance, including the ability to categorize emotionally significant information. The aim of our study was to analyze the recognition of emotional and neutral visual scenes in carriers of different polymorphic variants of the MAOA, COMT, DRD4, and 5HT2A genes. The study sample consisted of 87 university students (Caucasians, women 63%, average age 20.4±2.6 years). The genotypes of the COMT, 5HT2A, and DRD4 genes were determined by polymerase chain reaction. Agarose gel electrophoresis was used to determine the number of tandem repeats of the MAOA gene. Three hundred sixty (360) photographic images of scenes of different emotional valence (positive, negative, and neutral - 120 images for each category) were used as stimuli. These images were classified by expert assessments. The images were presented in a random sequence. The exposure time was 700 ms. The research participants were asked to determine the emotional valence of each scene. We found that only the COMT gene genotype affected the recognition of emotional and neutral visual scenes. Carriers of the COMT Val/Val genotype, which causes dopamine to stay in the synaptic space for a shorter time, are better in recognizing and demonstrate higher sensitivity to the emotional content of scenes. Carriers of the Val/Met genotype demonstrated the worst ability to differentiate the emotional valence of visual scenes. This study has shown that the length of stay of monoamines in the synaptic space regulated by the COMT gene affects the recognition of emotional visual information.

A summary-statistics-based approach to examine the role of serotonin transporter promoter tandem repeat polymorphism in psychiatric phenotypes

In genetic studies of psychiatric disorders in the pre-genome-wide association study (GWAS) era, one of the most commonly studied loci is the serotonin transporter (SLC6A4) promoter polymorphism, a 43-base-pair insertion/deletion polymorphism in the promoter region (5-HTTLPR). The genetic association signals between 5-HTTLPR and psychiatric phenotypes, however, have been inconsistent across many studies. Since the polymorphism cannot be tested via available SNP arrays, we had previously proposed an efficient machine learning algorithm to predict the genotypes of 5-HTTLPR based on the genotypes of eight nearby SNPs, which requires access to individual-level genotype and phenotype data. To utilize the advantage of publicly available GWAS summary statistics obtained from studies with very large sample sizes, we develop a GWAS summary-statistics-based approach for testing the variable number of tandem repeat (VNTR) associations with various phenotypes. We first cross-verify the accuracy of the summary-statistics-based approach for 61 phenotypes in the UK Biobank. Since we observed a strong similarity between the predicted individual-level 5-HTTLPR genotype-based approach and the summary-statistics-based approach, we applied our method to the available neurobehavioral GWAS summary statistics data obtained from large-scale GWAS. We found no genome-wide significant evidence for association between 5-HTTLPR and any of the neurobehavioral traits. We did observe, however, genome-wide significant evidence for association between this locus and human adult height, BMI, and total cholesterol. Our summary-statistics-based approach provides a systematic way to examine the role of VNTRs and related types of genetic polymorphisms in disease risk and trait susceptibility of phenotypes for which large-scale GWAS summary statistics data are available.

VNTR polymorphism in the SLC6A3 gene does not influence dopamine transporter availability measured by [18F]FE-PE2I PET or [123I]FP-Cit SPECT.

To investigate the potential impact of polymorphism in the 3'-untranslated region (3'UTR) of the SLC6A3 gene (DAT1) on normal variation in dopamine transporter (DAT) imaging with [18F]FE-PE2I PET and [123I]FP-Cit SPECT. Thirty-six individuals (mean age 70.4±5.4 years) with normal [18F]FE-PE2I PET and [123I]FP-Cit SPECT were genotyped for variable number of tandem repeats (VNTR) in the 3'UTR of the DAT1 gene. The DAT-availability in the caudate and putamen as measured with [18F]FE-PE2I PET and [123I]FP-Cit SPECT, as well as in the substantia nigra with [18F]FE-PE2I PET were compared between the participants carrying one or two 9-repeat alleles (i.e. 9R+10R or 9R+9R; 47%) and the participants without a 9R allele (i.e. 10R+10R or 10R+11R; 53%). Nonparametric tests, linear regression analysis and mixed model analysis were used to assess any statistical difference in measured DAT availability between the two allele groups. The measured DAT-availability in PET- and SPECT-imaging tended to be slightly higher in the 9R-group; however, the difference did not reach statistical significance in either the caudate or the putamen or the substantia nigra. Instead, age did have a significant effect on the DAT level (P < 0.05) notwithstanding the genotype. No significant effect of DAT1-genotype was detectable in imaging with [18F]FE-PE2I PET or [123I]FP-Cit, instead, age accounted for the normal variation in DAT-PET and DAT-SPECT.

Childhood trauma and genetic variation in the DAT 40-bp VNTR contribute to HIV-associated neurocognitive disorders.

HIV/AIDS is a major public health burden in South Africa, currently affecting an estimated 13.5% of the population. Despite improved access to antiretroviral therapies, HIV-associated neurocognitive disorders (HAND), characterised by a spectrum of neurocognitive impairment, emotional disturbances and motor abnormalities, continue to persist. Gene-environment interactions contribute to HAND pathophysiology and previous research has identified childhood trauma as an environmental risk factor. Dopaminergic signalling in the prefrontal cortex plays a key role in cognitive function. Thus, variants in genes encoding the dopamine transporter (DAT) and catechol-O-methyltransferase (COMT), which are responsible for dopamine transport and metabolism, could represent genetic risk factors for HAND. This study investigated whether the DAT variable number of tandem repeats (VNTR) and COMT Val158Met (rs4680) polymorphisms are associated with longitudinal change in cognitive function in the context of childhood trauma and HIV. Participants (n = 49 HIV-negative and n = 64 HIV-positive women) completed the Childhood Trauma Questionnaire – Short Form (CTQ-SF) and provided blood for genetic analyses. Global cognitive scores were generated from baseline and one-year follow-up assessments. Following polymerase chain reaction, genotypes were determined using gel electrophoresis and confirmed by Sanger sequencing. Baseline global cognitive scores, genotype, HIV status and CTQ-SF scores were regressed on one-year global cognitive scores in regression models. Analysis of variance was used to examine the effect of including predictor variable interactions on model fit. HIV seropositivity was associated with poorer cognitive performance at one-year follow-up (p = 2.46 ×10−4). The combination of HIV and DAT 10-repeat homozygosity (DAT 10/10) was associated with reduced global cognitive scores in longitudinal models (p = 0.010). Including the interaction between DAT 10/10, childhood trauma, and HIV explained significantly more of the variance in longitudinal cognitive scores (p = 0.008). There were no significant associations with the COMT genotype. Our research indicates that childhood trauma and genetic variation in DAT contribute toward the aetiology of HAND. Future studies in larger cohorts are warranted to verify these results.

Bacillus anthracis Phylogeography: New Clues From Kazakhstan, Central Asia

This article describes Bacillus anthracis strains isolated in Kazakhstan since the 1950s until year 2016 from sixty-one independent events associated with anthrax in humans and animals. One hundred and fifty-four strains were first genotyped by Multiple Locus VNTR (variable number of tandem repeats) Analysis (MLVA) using 31 VNTR loci. Thirty-five MLVA31 genotypes were resolved, 28 belong to the A1/TEA group, five to A3/Sterne-Ames group, one to A4/Vollum and one to the B clade. This is the first report of the presence of the B-clade in Kazakhstan. The MLVA31 results and epidemiological data were combined to select a subset of seventy-nine representative strains for draft whole genome sequencing (WGS). Strains from Kazakhstan significantly enrich the known phylogeny of the Ames group polytomy, including the description of a new branch closest to the Texas, United States A.Br.Ames sublineage stricto sensu. Three among the seven currently defined branches in the TEA polytomy are present in Kazakhstan, “Tsiankovskii”, “Heroin”, and “Sanitary Technical Institute (STI)”. In particular, strains from the STI lineage are largely predominant in Kazakhstan and introduce numerous deep branching STI sublineages, demonstrating a high geographic correspondence between “STI” and Kazakhstan, Central Asia. This observation is a strong indication that the TEA polytomy emerged after the last political unification of Asian steppes in the fourteenth century of the Common Era. The phylogenetic analysis of the Kazakhstan data and of currently available WGS data of worldwide origin strengthens our understanding of B. anthracis geographic expansions in the past seven centuries.

Two New Mutations in the CEL Gene Causing Diabetes and Hereditary Pancreatitis: How to Correctly Identify MODY8 Cases

ContextMaturity onset diabetes of the young, type 8 (MODY8) is associated with mutations in the CEL gene, which encodes the digestive enzyme carboxyl ester lipase. Several diabetes cases and families have in recent years been attributed to mutations in CEL without any functional or clinical evidence provided.ObjectiveTo facilitate correct MODY8 diagnostics, we screened 2 cohorts of diabetes patients and delineated the phenotype.MethodsYoung, lean Swedish and Finnish patients with a diagnosis of type 2 diabetes (352 cases, 406 controls) were screened for mutations in the CEL gene. We also screened 58 Czech MODY cases who had tested negative for common MODY genes. For CEL mutation-positive subjects, family history was recorded, and clinical investigations and pancreatic imaging performed.ResultsTwo cases (1 Swedish and 1 Czech) with germline mutation in CEL were identified. Clinical and radiological investigations of these 2 probands and their families revealed dominantly inherited insulin-dependent diabetes, pancreatic exocrine dysfunction, and atrophic pancreas with lipomatosis and cysts. Notably, hereditary pancreatitis was the predominant phenotype in 1 pedigree. Both families carried single-base pair deletions in the proximal part of the CEL variable number of tandem repeat (VNTR) region in exon 11. The mutations are predicted to lead to aberrant protein tails that make the CEL protein susceptible to aggregation.ConclusionThe diagnosis of MODY8 requires a pancreatic exocrine phenotype and a deletion in the CEL VNTR in addition to dominantly inherited diabetes. CEL screening may be warranted also in families with hereditary pancreatitis of unknown genetic etiology.

Primer Designing for Amplifying an AT-Rich Promoter from Arabidopsis thaliana.

The aim of the present study is to optimize the PCR conditions required to amplify the promoter sequence of an amino acid transporter having an AT-rich base composition with a high number of tandem repeats. The present studyalsocovers the key parameters that need to be kept in mind while designing primers. Results show that successful can be achieved by performing a 2-step PCR reaction at a lower extension temperature of 65 ̊C for an increased extension period of 1.5 min/kb, with MgCl2 concentration ranging from 2.5 to 3.0mM. The results also suggest that the DNA concentration of around 25-30 ng/µl was essential to achieve this amplification.