Articles

Can genomics enable genetic evaluations with phenotypes recorded on smallholder dairy farms?


Identification: Powell, Owen

Credits: None available.

Can genomics enable genetic evaluations with phenotypes recorded on smallholder dairy farms?
 
Owen Powell1, R. Chris Gaynor1, Janez Jenko1, Gregor Gorjanc1, Okeyo Mwai2, Raphael Mrode2,3 & John M. Hickey1
1The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Research Centre, Midlothian EH25 9RG, UK; 2ILRI, International Livestock Research Institute; 3Scotland's Rural College (SRUC), Peter Wilson Building, Kings Buildings, West Mains Road, Edinburgh, EH9 3JG
 
Background: Genetic evaluation is a central component of genetic improvement programs. In advanced economies, most genetic evaluations depend on large quantities of data that are recorded on commercial farms. Large herd sizes and widespread use of artificial insemination enable the genetic and environmental components of an individual animal's phenotype to be accurately separated. In contrast to this, herds are neither large nor have high genetic connectedness in smallholder farming systems, such as in East Africa. This limits genetic evaluation with pedigree information. Genomic information keeps track of shared haplotypes rather than animals. This information could capture and strengthen connectedness between herds and through this may enable genetic evaluations based on phenotypes recorded on smallholder dairy farms. The objective of this study was to use simulation to quantify the power of genomic information to enable genetic evaluation under such conditions.
 
Results: The results from this study show; (i) GBLUP produced higher accuracies than PBLUP at all population sizes and herd sizes, (ii) Models with herd fitted as a random effect produced equal or higher accuracies than the model with herd fitted as a fixed effect across all herd size scenarios, (iii) At low levels of genetic connectedness, with four offspring per sire and one to two animals per herd, GBLUP produced EBV accuracies greater than 0.5. Generally, a decrease in the number of sires mated per generation showed consistently higher accuracies compared to when more sires were used.
 
Conclusions: This study has demonstrated the potential of genomic information to be an enabling technology in smallholder dairy economies by facilitating genetic evaluations with records collected from farms with herd sizes of four cows or less.  The inclusion of smallholder dairy data in genetic evaluations could provide increases in local and national milk production, in regions such as East Africa, with downstream impacts upon wider societal, nutritional and economic outcomes.
 

First molecular detection of pathogenic Leptospira in livestock from slaughterhouses in Madagascar


Identification: Rahelinirina, Soanandrasana

Credits: None available.

First molecular detection of pathogenic Leptospira in livestock from slaughterhouses in Madagascar
 
Soanandrasana Rahelinirina1, Mark Moseley2, Minoarisoa Rajerison1, Rakotoharinome Vincent Michel3, Sandra Telfer1,2
1Institut Pasteur de Madagascar, 2The University of Aberdeen, 3Direction des Services Vétérinaires Madagascar
 
Although leptospirosis has not been considered a significant health problem for humans or livestock in Madagascar, a recent serological study has shown evidence of exposure in humans and identified contact with cattle as a key risk factor.  However, the real role of livestock such as cattle and pig in the epidemiology of human leptospirosis in Madagascar remains to be elucidated. The aims of this study were to determine the prevalence of Leptospira among livestock in Madagascar and characterize the pathogenic Leptospira species found. Kidney and urine samples from 105 cattle and 100 pigs were collected from four slaughterhouses in Madagascar in 2015. The samples were tested by a quantitative real-time PCR TaqMan assay targeting the 16sRNA gene specific to pathogenic Leptospira. Where possible, sequencing of 200-300bp of the lfb1 gene was used to identify infecting Leptospira species. Cattle had a significantly higher overall prevalence than pigs (19% vs 5%). Cattle had 13 infections detected in kidney samples and 11 in urine samples, with only 4 individuals testing positive for both sample types. Of the positive pigs, only one infection was detected in kidney compared to four infections detected in urine.
Sequencing identified L. borgpetersenii and L. kirschneri in cattle. No lfb1 sequences were obtained from positive pig samples. This is the first molecular evidence of leptospirosis in livestock in Madagascar. The high prevalence in cattle provides further evidence that they could be an important source of infection for humans and also suggest that leptospirosis may have significant impacts on animal health and productivity. Moreover, our results highlight the need to consider sampling methodology when comparing studies and planning surveillance. Our results reinforce the need to take a one health approach for zoonoses like leptospirosis.
 

Genomic Characterization of Zambian Indigenous Cattle Breeds


Identification: Smith, Edward

Credits: None available.

 
Genomic Characterization of Zambian Indigenous Cattle Breeds
 
Edward Smith
Virginia Polytechnic Institute and State University, Animal and Poultry Sciences, Blacksburg, VA
 
Breed characterization is a primary step in designing appropriate management and conservation programs of livestock in developing countries. Since cattle represent a major food animal species in Zambia, its conservation is a major goal for both the government and non-governmental organizations. To support the conservation effort, the objective of this thesis research was to assess the phenotypic and molecular characteristics of indigenous Zambian cattle breeds including Angoni, Barotse, Tonga, and Baila based on body measurements and randomly amplified polymorphic DNA (RAPD) markers, respectively. A total of 100 animals, 25 from each of the four breeds associated with different tribes and region of Zambia, were used in the molecular analysis research. Additionally, 10 Holstein x Jersey crossbred animals were used as a reference and to test the extent of cross-breeding, if any, of the indigenous stock with exotic breeds. To further compare the Zambian indigenous breeds, morphometric measurements including body length, heart girth, and height at withers on 50 animals of each breed were measured. Blood was collected from animals at randomly selected farms and DNA isolated by standard protocols in Zambia. A total of 10 primers, of the 20 evaluated for informativeness, were used in the RAPD-PCR analyses. Differences among the four breeds for all the three morphometric measurements were significant with the Barotse significantly higher than the other three (P<0.05). The average number of bands per primer was 7.1 and the percentage of polymorphic bands per primer ranged from 40 to 71.4 with an average of 64.8%.  Breed divergence was highest between the Tonga and the Barotse and lowest between the Tonga and Baila breeds.  Both the morphometric measurements and RAPD-based distance estimates suggest that the Barotse may be different from the other indigenous breeds while the Tonga and Baila were more closely related. In addition, the genetic distance estimates imply that the Holstein x Jersey crosses are different from the four Zambian indigenous cattle breeds evaluated. This thesis research provides, for the first time, the basic genetic information necessary for conservation of Zambian cattle breeds and the use of these populations for effective crossbreeding. The data suggest that though there is isolated by geographic distance and cultural differences among the tribes, two of the breeds are significantly related.
 
 

Genetic diversity of pharmacogenes in a Bantu-speaking cohort and evaluation of variants associated with tenofovir-induced nephrotoxicity


Identification: Tshabalala, Elizabeth

Credits: None available.

Genetic diversity of pharmacogenes in a Bantu-speaking cohort and evaluation of variants associated with tenofovir-induced nephrotoxicity
 
E. Sibongile Tshabalala1, Ananyo Choudhury2, Natasha Beeton-Kempen1, Faheem Seedat3 and Ebrahim Variava3, Neil Martinson4, Michèle Ramsay2, 5 and Dalu Mancama1
1CSIR, Biosciences Unit, South Africa: 2Sydney Brenner Institute for Molecular Bioscience, Faculty of Health Sciences, University of the Witwatersrand, South Africa; 3Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 4Perinatal HIV Research Unit, Baragwanath Hospital and Faculty of Health Sciences, University of the Witwatersrand, South Africa; 5Division of Human Genetics, National Health Laboratory Service, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, South Africa
 
Recent South African studies report an increase in hospital admissions due to adverse drug reactions (ADRs), including to tenofovir disoproxil fumarate (TDF). Growing evidence suggests a genetic contribution to TDF-related ADRs. Given the high genetic diversity observed in African populations, the aim of this study was to elucidate the pharmacological implications of such diversity by identifying and characterizing known and novel pharmacogenetic variants and evaluating their possible association with TDF-induced nephrotoxicity. Using targeted next generation sequencing to screen 40 Bantu-speaking individuals for variants in 65 genes, 1687 variants were identified; including 129 novel and 22 potential loss-of-function variants. Based on allele frequency (MAF>0.1) and prior association with ADRs, nine SNPs within five genes were prioritised for a genetic association study for TDF-induced nephrotoxicity (clinically manifesting as acute kidney injury (AKI)).  A total of 137 HIV positive patients on TDF treatment were subsequently genotyped using TaqMan® assays, 53 of whom presented with AKI. Association analysis was performed with alleles, genotypes and haplotypes using χ2 tests. The ABCC2 1249A allele and ABCC2 haplotypes AAC and AAT displayed associations with TDF-induced AKI (p≤0.05). The ABCC2 GTT haplotype (p=0.02) appeared to be protective against TDF-induced AKI. However, the associations were not significant following corrections for multiple testing. Further evaluation of these ABCC2 variants in larger cohorts is warranted to establish their role, if any, in TDF-induced AKI.
 

Non-typhoidal Salmonella and Zoonotic E. coli as Potential Drivers of Antimicrobial Resistance in Pastoralist Communities in Kasese District, Uganda


Identification: Walusansa, Abdul

Credits: None available.

Non-typhoidal Salmonella and Zoonotic E. coli as Potential Drivers of Antimicrobial Resistance in Pastoralist Communities in Kasese District, Uganda
 
Abdul Walusansa
School of Medicine, Islamic University in Uganda

August 2017
 
Introduction: Non-prescribed use of antimicrobials in Agriculture incurs a transfer risk of resistant pathogens to humans. The aim of this study was to determine the potential of non-typhoidal Salmonella spp and Zoonotic E. coli to serve as drivers of Antimicrobial resistance among animals and humans.
 
Methodology: A laboratory based cross-sectional study was done using archived Salmonella spp and E. coli isolates previously obtained from individuals among pastoralist communities of Kasese district, Uganda. Recovery of the isolates was done by conventional culture and Identification by biochemical methods, serotyping and PCR. Antimicrobial resistance profiling was done by using Kirby bauer disc diffusion method. Following this; the isolates were screened for resistance mechanisms including Extended Spectrum β-lactamase, Carbapenemase and AmpC production using disc diffusion based methods.
 
Results: The prevalence of Enterohemorrhagic E.coli (EHEC) and Non Typhoidal salmonella were 16% (28/180) and 50% (1/2) respectively. Of the EHEC, 94% (26/28) were of phylogroup B1, A (3%, 1/28) and B2 (3%, 1/28). The most prevalent virulence gene in the EHEC was Stx1 (100%, 28/28) followed by Stx2e (94%, 26/28), none was Stx2 positive. Highest resistance was seen to Cotrimoxazole (89%, 25/28), Tetracycline (71%,20/28), Ampicillin (65%,18/28) and Nitrofurantoin (28%,8/28), these are commonly used in the agricultural sector, whereas minimal resistance was observed to those commonly used in human medicine especially the β-lactams, β-lactam inhibitors and Carbapenems. 17%, (5/28) of the EHEC were ESBL positive, of these one (3%, 1/28) was a Carbapenemase producer. Though only one Non-typhoidal Salmonella isolate was found, it is worrying to note that it showed resistance to all the three antimicrobial agents (Nalidixic acid, Chloramphenicol and Ciprofloxacin) that are most recommended for treatment of Salmonellosis in this setting.
 
Conclusion: There is a high prevalence of highly pathogenic and resistant zoonotic E. coli and low prevalence of Non typhoidal salmonella among humans in pastoralist communities in Uganda. We suspect that these pathogens, along with their AMR genes, were acquired from animals because the zoonotic E. coli (EHEC) largely contained the animal specific Vero toxin gene VT2e and majority belonged Pylo-group B1 which has been documented as the most common EHEC phylo-group inhabiting domestic animals. Therefore, it is highly likely that zoonotic bacteria are potential drivers of antimicrobial resistance to humans in these settings and we recommend that studies involving relatedness of drug resistant isolates from humans and animals should be conducted to ascertain the role of enterohemorrhagic E. coli in the zoonotic spread of antimicrobial resistance in pastoralist communities. We recommend that a one health approach should be used to establish drivers of MDR spread in pastoralist communities.
 

High Heterozygosity in SNPs of TNF alpha Gene of Women Accessing Breast Cancer Care at a Tertiary Health Facility in Southwest Nigeria


Identification: Alamukii, Nanfizat Abiket

Credits: None available.

High Heterozygosity in SNPs of TNF alpha Gene of Women Accessing Breast Cancer Care at a Tertiary Health Facility in Southwest Nigeria
 
Nanfizat Abiket Alamukii1,2*, Abayomi Odetunde2, Chinedum Peace Babalola2,,3 Adeyinka Gladys Falusi2, Roseangela Ifeyinwa Nwuba1,4
1
Department of Zoology, Faculty of Science, University of Ibadan, Nigeria; 2Genetics Research Unit, Institute for Advance Medical Research and Training (IAMRAT), University College Hospital (UCH), University of Ibadan, Nigeria; 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ibadan, Nigeria; 4Department of Biological Sciences, Faculty of science, University of Medical Sciences, Ondo, Nigeria
*Presenting author

 
Introduction: Single Nucleotide Polymorphisms (SNPs) at the promoter region of TNFα gene are involved in the regulation of its expression. Alleles of these SNPs such as TNFα 308-A have been known to increase the production TNF protein. The presence of TNFα at the microevironment of certain tumours including breast cancer have been recorded. We genotyped the SNPs of TNFα and its receptors in breast cancer patients and control individuals to determine their role in the development and progression of breast cancer among women accessing care at a tertiary health facility in southwest Nigeria.
 
Methodology: This is a case control study of 200 consenting women recruited from the University College Hospital, Ibadan. The study was approved by the UI/UCH Ethical Review Board/Committee. DNA was extracted from blood samples collected from each participant. Allele-specific polymerase chain reaction was used to genotype various SNPS of TNFα and its receptors [TNFα (488 G/A, 238 G/A, 308 G/A, 859 C/T) and its receptor (TNFR1A+IV56+10 -G/A)].
 
Results: These showed very high heterozygosity of all the TNFα SNPs genotyped in the population which is a deviation from Hardy-Weinberg rule (P<0.0001). There was a significant difference (p-=0.0213) between cases and control for genotypes GA, AA & GG of TNFα 308. On the other hand, there were no significant differences between case and control for genotypes GA, GG & AA of TNFα 488, 238, 380 TNFR1A+IV56+10, and CC, CT, TT of TNFα 859 (P=0.2841, 0.7859, 0.3014 & 0.4922 respectively).
 
Conclusion: The genotypic results indicate a selection for heterozygote alleles within the population which although not yet linked to breast cancer, may be important in the expression of TNF gene for survival the geographical region. The significant difference in the genotypes of TNFα 308 between breast cancer patients and control individuals is a possible indicator that this SNP may have an important role in the development and progression of breast cancer among Nigerian women.
 
Keywords: Breast cancer, TNFα, SNPs heterozygosity, Nigerian women.
 
 
 

Routine use of targeted NGS panel in a Dutch cardiomyopathy cohort


Identification: Alimohamed, Mohamed

Credits: None available.

Routine use of targeted NGS panel in a Dutch cardiomyopathy cohort
 
Mohamed Z. Alimohamed, Lennart F. Johansson, Ludolf G. Boven, Krista van Dijk, Paul van der Zwaag, Richard J. Sinke, Rolf H. Sijmons, Birgit Sikkema-Raddatz, Jan D H Jongbloed
Departments of Genetics, University Medical Center Groningen, Groningen, The Netherlands
 
Background:
Next generation sequencing is increasingly used for clinical evaluation of patients with cardiomyopathies because it allows for simultaneous evaluation of multiple genes known to be associated with the disease. However, the diagnostic yield is variable in routine clinical practice. Furthermore, analysis of copy number variations is still not routinely performed.
 
Objectives:
(1) To determine the diagnostic yield of our custom targeted NGS gene panel used in routine clinical diagnostics in a Dutch cohort of over 2000 cardiomyopathy patients. (2) Examine the impact on the yield of analyzing this cohort for single or multiple exon duplications and deletions.
 
Methods:
Up to 61 genes known to be implicated in cardiomyopathies were selected for enrichment and analysis on DNA isolated from peripheral blood from patients as a routine procedure. Patients directed for genetic testing to our clinical genetics laboratory having a referral diagnosis for various types of cardiomyopathies were included in this study [N=2002]. A written informed consent was obtained for all patients referred to our clinical genetics laboratory. Classification of variants was based on guidelines for variant interpretation recommended by the American College of Medical Genetics and Genomics. Diagnostic yield was calculated using cases where a likely pathogenic or pathogenic variant was detected. Putative exonic deletions/duplications were analyzed using CoNVaDING and XHMM tools using settings previously described.
 
Results and Conclusion:
An overall diagnostic yield of 23% was achieved. CNVs were detected in 16 patients. Variants of unknown clinical significance were identified in 39% patients. These results illustrate the need to further reassess disease variant classification.
 
Key words:
Cardiomyopathy, NGS-panel, Diagnostic-yield

Evaluation of Genetic Test Screening on Epidemiology of Prostate Cancer in Swaziland: A Systematic Study of Mutated Genes and Causal Factors


Identification: Fashoto, Stephen

Credits: None available.

Evaluation of Genetic Test Screening on Epidemiology of Prostate Cancer in Swaziland: A Systematic Study of Mutated Genes and Causal Factors
 
Fashoto, S.G.1, Akinnuwesi, B. A.2, & Owolabi, O.3
1Department of Computer Science University of Swaziland, Kwaluseni, Swaziland; 2Department of Computer Science, Lagos State University, Ojo, Lagos, Nigeria; 3Department of Computer Science, University of Abuja, Abuja, Nigeria
 
In Swaziland, the annual mortality rate per 100,000 people due to Prostate Cancer (PCa) increases at an average of 2.6% per year. One of the major reasons for this is the difficulty of PCa diagnosis at the early stages because, in many cases, its mutated genes are confused with the mutated genes of some other diseases. For example, to assess an individual's chance of having prostate cancer, a genetic test is carried out to see if there are any mutated genes such as HOXB13, BRCA1 and BRCA2, although BRCA1 and BRCA2 are also implicated in ovarian and breast cancer. Genetic testing for prostate cancer helps in screening to determine any molecular mutation that might be an indicator for the possible development of prostate cancer. This study will investigate the use of machine learning techniques to replace the most commonly used genetic test called prostate specific antigen (PSA), which is generally  considered to be unreliable, to determine if the probability of an individual developing PCa could be detected early enough for possible management and treatment. We propose to investigate epidemiological status of PCa, with the view to establishing the PCa types, vis-à-vis the primary and secondary factors responsible for PCa cases in Swaziland. This study will also attempt to identify the symptoms, vis-à-vis the confusability of the mutated genes with other diseases and the medical diagnostic procedures adopted.
 
 

Genetic Diversity of Plasmodium falciparum Parasites in Pregnant and Non-pregnant Women and Potential Resistance to Antimalarial Drugs in Kenya


Identification: Makena, Brenda

Credits: None available.

Genetic Diversity of Plasmodium falciparum Parasites in Pregnant and Non-pregnant Women and Potential Resistance to Antimalarial Drugs in Kenya
 
Brenda Makena1,2, Martha Nginya1,2, Marcel Nyambute1, Charles Okello1, Edwin Mwakio1, Gladys Chemwor1, Raphael Okoth1, Redemptah Yeda1, Agnes Cheruiyot1, Benjamin Opot1, Dennis Juma1, Victor Mobegi2, Hosea Akala1, Ben Andagalu1
1United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)
2Centre for Biotechnology and Bioinformatics (CEBIB). University of Nairobi, Nairobi, Kenya.
 
During pregnancy, malaria causes maternal anaemia, placental accumulation of parasites, low birth weight, infant mortality as well as maternal mortality. Pregnant women are presumed non-immune to malaria whereas the non-pregnant are thought to be semi-immune. Non-pregnant women therefore have faster parasite clearance compared to pregnant women. The non-immune environment in pregnant women may select for parasite populations that are associated with resistance to artemisinin. This study will determine genetic variations in Plasmodium falciparum parasites in pregnant versus non-pregnant women due to differences in their immunity. Samples were collected at hours 0, 8, 24 and at days 7 and 28 from 75 participants in an efficacy study. Rapid diagnostic test and microscopy will be done followed by 18s rRNA PCR for speciation analysis. Sanger sequencing and MassARRAY SNP genotyping will be done to determine mutations in parasites without fast clearance as well as microsatellite genotyping to determine genetic variations in the parasites between the two populations. Genotyping for both MSP1 and MSP2 will then follow to determine recrudescence and reinfection. Twelve microsatellite markers will be used to calculate allele frequencies, heterozygosity (He) values, molecular variance, haplotypes, principal coordinates and fixation indices using GenAIEx and Arlequin softwares. For MassARRAY SNP genotyping, genotype calls will be made using SpectroTyper 4.0 software (Agena). For the K13 and Sanger sequencing SNPs will be analyzed by doing multiple sequence alignment using CLC Genomics Workbench software. This study will contribute to knowledge on guiding the World Health Organization implementation of intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine if found to influence parasites response to artemisinins.
Funding: Armed Forces Health Surveillance Branch (AFHSB) and Developing Excellence in Leadership and Genetics Training for Malaria Elimination in Sub-Africa (DELGEME)
 

Surveillance of pre-treatment drug resistance among HIV infected children in Ibadan, Nigeria


Identification: Olusola, Fiyinfoluwa

Credits: None available.

Surveillance of pre-treatment drug resistance among HIV infected children in Ibadan, Nigeria
 
Olusola Fiyinfoluwa I1,*, Oladokun Regina2, Falade Catherine1,3
1Department of Pharmacology and therapeutics, University of Ibadan, Ibadan.2Department of Paediatrics, College of Medicine, University of Ibadan & University College Hospital, Ibadan; 3Institute for Medical Research and training, University of Ibadan, Ibadan
 
There are about 2.1 million children infected with HIV globally out of which 120 thousand die annually. Nigeria has the highest rate of paediatric HIV infection globally. Despite this, there is limited information on the burden of HIV-1 drug resistance among children in the country. The emergence of drug resistant strains is a significant contributor to morbidity and mortality due to HIV infection. Pre-treatment HIV drug resistance data inform the choice of first- and second-line antiretroviral therapy (ART) regimens.  This study therefore investigated the prevalence of HIV-1 drug resistant strains among ART naïve children in Ibadan, Nigeria.            A total of 20 children aged less than 15 years were enrolled.  Demographic, clinical and laboratory data were documented. Total nucleic acid was extracted from blood samples after which amplification of HIV-1 pol gene was done using polymerase chain reaction. Amplified gene was sequenced using big dye sequencing method. The sequenced HIV-1 pol genes were typed and analysed for identification of mutations indicative of drug resistance across the different classes of antiretroviral therapy using Rega subtyping tool Version 3 and Stanford HIV Drug Resistance Database. HIV 1 RNA pol gene was successfully amplified and identified in 12/20 (60%) children.  These sequences were identified as HIV-1 subtypes G (n=4; 33%), CRF 02AG (n=4; 33%), Recombinant AG/G (1(8.3) and Recombinant AG/A1 (1(8.3).  Two sequences (16.6%) were untypable. Drug resistant mutations were identified in four samples (33%), out of which three were NNRTI DRM (K103N) and the fourth a NRTI DRM (M184V). Results from this preliminary study suggest that HIV-1 drug resistance among ART-naïve children is a problem in Ibadan, Nigeria.   Pre-treatment drug resistant testing is desirable in children prior to initiation of ART to guide effective treatment.