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Next Generation Sequencing in the field of Human Genetics
Author: Alvera Vorster & Carel van Heerden
Published: 02/10/2018

The DNA sequencing unit has a long history of supporting research at Stellenbosch University, as well as other universities. Since its inception over two decades ago, the unit has supported research in the field of Human Genetics though cycle sequencing and fragment analysis.

This traditional service provision role was expanded to include next generation sequencing (NGS), following a National Equipment Program grant award to Prof Johan Burger; for the procurement of the ABI 5500xl and Ion Torrent Personal Genome Machine (PGM). With these instruments in its arsenal, the unit aimed to become a trusted provider of high quality massively parallel sequencing data.

In September 2013 the DNA sequencing unit became the first laboratory to sequence a human genome on African soil, using the 5500xl. With the 5500xl dedicated to the generation of large datasets, the PGM was employed to generate data for small genomes or sub-sets of larger genomes through fragmentation-based workflows. The introduction of the targeted sequencing technologies like AmpliSeq TM soon afterwards allowed the PGM to be employed to investigate specific areas of the human genome at a reduced cost and with an improved data delivery time.

A second NEP grant was awarded to Prof Altus Viljoen for the procurement of an Ion Torrent Proton. This platform had approximately 10x the data capacity of the PGM and led to the birth of the whole human exome (WES) sequencing service that we are currently offering. To date, the Ion Proton has generated 326 data sets – approximately 4 890 000 000 000 000 bp of data.

This includes more than 280 human exomes; half of which were sequenced in 2017/2018. These WES projects, some small scale (n = 2) and other large scale (n = 100), are co-ordinated by South Africans, to investigate the genetic determinants of a range of heritable human disorders in the local context.

These are four of the research groups in the field of Human Genetics that are currently supported by the NGS service; Cardiovascular Genetics Group at the University of Cape Town. The Cardiovascular Genetics Group ( aims to discover the genetic causes of inherited and sporadic heart diseases in South Africa. By studying families with rare monogenic diseases, they hope to identify the mutations and biological pathways that ultimately may be targeted to relieve symptoms and prevent sudden cardiac death in patients. In order to address this aim, they have turned to next generation sequencing, specifically whole exome sequencing (WES), as the preferred method of DNA variant detection.

During the first six months of 2018, 100 genomic samples were submitted to the DNA sequencing unit at the CAF for WES. This data that had been generated, to support the research efforts of the Cardiovascular Genetics Group, are the first exomes for congenital heart disease patients in Africa.

"I have been very impressed with how the projects were handled, from start to finish. At the start of each project I would receive an expected time of completion of the exomes and when the data would be available to me as the client (via Ion Reporter and/or external hard drive). During the actual process I would receive regular updates on what stage the process was at and if any problems had been encountered. I appreciated this most of all [sic] as this allowed me to plan around the issues (not that there were many). We are still in the early stages of the data analysis but I have been very impressed with the amount of data that they generated, their data quality as well as their service delivery times; that was spot on. They have been professional, efficient and if the way they handled this project is any indication we should be having some great genetics results for heart disease soon."

– Dr Gasnat Shaboodien – Deputy Director, Cardiovascular Genetics Research Group, Hatter institute for Cardiovascular research in Africa

The Parkinson’s disease Research Group at Stellenbosch University

The Parkinson’s disease (PD) Research Group at Stellenbosch University ( focuses on studying the genetic basis in South African patients with PD, as well as the underlying disease mechanisms. PD is the most common neurodegenerative movement disorder and results from the loss of neuronal cells in a specific part of the brain known as the substantia nigra. Symptoms include slow movements and reflexes, involuntary trembling of the body and limbs, stiff muscles, difficulty in maintaining balance, and patients also experience various psychological manifestations.

The prevalence of PD in South Africa is not known but it is estimated to affect seven to ten million people worldwide. The advent of high-throughput NGS technologies has revolutionised the way patient’s genomes can be screened for genetic defects that can lead to PD. Although these approaches are quite expensive, in the long run, they are more cost-effective than the traditional methods used in the past. Currently, NGS is used in two student research projects.

Mr Oluwafemi Oluwole is pursuing doctoral studies and his project involves the use of a commercially available targeted resequencing gene panel from Thermo Fisher Scientific. In collaboration with a team in Nigeria, PD patients from both countries are screened for genetic defects in 751 genes using a Neurological Research Gene panel on the Ion Torrent platform at CAF. Since PD has been significantly understudied in Black populations worldwide, this research is important and relevant.

To date, 33 black South African and 43 Nigerian PD patients have been sequenced and analysis of the results are underway. Preliminary analysis of data from 47 patients, show sequencing reads of good quality and after applying stringent quality criteria, a total of 2,021 variants were identified. Thirty rare variants, predicted to be pathogenic based on bioinformatic tools, were prioritised and will be studied to assess their involvement with the development of PD. These variants will be validated by direct cycle sequencing at CAF.

Ms Amokelani Mahungu is a Masters student and her project involves setting up a custom-designed gene panel for PD in order to facilitate the rapid screening of all the known PD-associated genes in a cohort of > 650 PD patients. To address this aim, a 23 gene panel was designed using Agilent’s SureSelect technology. This gene panel is due for evaluation during July 2018, when 16 selected PD patients will be sequenced on CAF’s Ion Torrent Proton platform. Given the unique ancestry of SA populations the PD research group anticipates that novel mutations in these genes may be identified.

"In summary, the NGS service that the DNA Sequencer Unit at CAF provides has had a major impact on our research. It allows us to comprehensively screen our unique collection of DNA samples obtained from South African patients. Ultimately, this has the potential to facilitate important breakthroughs on the genetic causes, in local patients, of this debilitating and poorly understood disorder."

Prof Soraya Bardien – Principle Investigator, PD Research Group at Stellenbosch University

(Image: Amica Muller-Nedebock)

Primary Immunodeficiency Diseases Genetics Network (PIDDGEN)

DST-NRF Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, NHLS Immunology Unit, Division Medical Microbiology, Department of Pathology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town.

Primary immunodeficiency diseases (PIDs) collectively represent a significant burden of disease. Recent estimates suggest that there are as many as 42,000 South African PID cases. Unfortunately the diagnosis of PIDs in our country have been hampered, in part, by high prevalence of several infectious diseases, lack of awareness and training and, despite available clinical algo­rithms and basic laboratory diag­nosis, the lack of transport and laboratory infrastructure for more advance immune test samples. To address these issues, the Primary Immunodeficiency Disorders Ge­ne­­tic Network (PIDDGEN), a multi­disciplinary team of researchers and clinicians from Stellenbosch University’s Faculty of Medicine and Health Sciences, have been providing molecular diagnoses to South African patients with Primary Immunodeficiencies.

Our patients have frequently seen many doctors and were subjected to many tests and futile treatments. Their journey through the health care system, "a diagnostic odyssey", frequently spans many years in search for an answer to the cause of their illness. PIDs, when left undiagnosed and untreated are often associated with severe morbidity and increased mortality. Quality of life for diagnosed PID patients is significantly improved compared to undiagnosed patients and there is a definite cost saving for diagnosed patients. However, for many PIDs, effective treatment is available, but since the diagnosis maybe quite challenging, several potentially treatable and serious PIDs remain undiagnosed. The PIDDGEN Program helps patients to access diagnostic genomics for previously undiagnosed diseases. All patients are carefully screened, counselled, and enrolled irrespective of financial or social background.

To facilitate molecular diagnoses, we have been using whole exome sequencing (WES) to identify the genetic mutations causing disease in each of our patients. The Stellenbosch University’s Central Analytical Facility has been our preferred WES service provider for a number of years and through this partnership, we have sequenced the exomes of 41 PID patients and 52 of their family members. Of these, we were thus far able to provide a molecular diagnosis for 27 (66%) of our PID patients. This is significant given that for several PIDs, the disease-causing mutation has a direct impact on the patient treatment.

Breast Cancer Research

Three major challenges in the field of breast cancer have been identified as research priorities. The first is the need to combine genetic testing of high-risk patients with familial breast cancer with pharmacogenetics to reduce recurrence risk in cancer survivors due to drug failure as a consequence of anti-cancer treatment that does not match the patient’s genotype. The second is the delineation of key pathways through which genes implicated in breast cancer and associated co-morbidities can serve as nutritional and drug targets across diagnostic boundaries.

The third is the discovery of genetic alterations underlying familial breast cancer not attributed to mutations in the two major tumour suppressor genes, BRCA1 and BRCA2 (see figure above).

There is currently no consensus on eligibility criteria for WES in BRCA1/2 mutation-negative familial breast cancer patients. To address these challenges, the pathology-supported genetic testing (PSGT) platform was used to develop an exome pre-screen algorithm (EPA) for selection of genetically uncharacterised patients for WES. First, diagnostic BRCA testing is offered as a routine service according to standard referral guidelines, or the chronic disease risk screen is offered to patients receiving hormone therapy, are at high risk for tumour recurrence, or are required to take potentially competing antidepressants.

In certain cases, combined diagnostic and pharma-cogenetics testing is performed to explain the presence of comorbidities or predict drug response/recurrence risk. Finally, where extended mutation analysis of the entire BRCA1 and 2 genes as well as the CYP2D6 gene is unable to explain breast cancer or the occurrence of drug side effects/failure, WES is performed to identify potential novel causative genes/mutations.

In a study that consisted of 164 breast cancer patients (60 Mixed Ancestry and 104 Caucasian) and 160 cancer-free controls, common genetic risk factors for cardiovascular disease (CVD) were shown to be significantly associated with earlier age (10 years on average) of breast cancer onset/diagnosis and body mass index (BMI) in patients stratified according to estrogen receptor (ER) status, after adjustment for potential confounders. Age at diagnosis/onset of breast cancer was significantly lower in patients with ER-negative versus ER-positive tumours, after adjustment for ethnicity, while BMI was significantly higher in patients with ER-positive compared to ER-negative tumours after adjustment for age, ethnicity, and a family history of cancer.

These findings contributed to the development of the EPA used to select a genetically uncharacterized family for WES and facilitated the development of a framework for WES performed alongside clinical and pathology assessments. Results supported previous findings indicating that the majority of genetically uncharacterised breast cancer cases may be caused by a combination of low–moderate penetrance mutations exerting their effect against a high-risk clinical background.

To our knowledge, this is the first study using WES to investigate the significance of folate pathway SNPs as risk reduction targets beyond BRCA1/2 in familial risk. WES preceded by PSGT facilitated the identification and clinical interpretation of genetic risk factors of relevance to both cancer development and tailored therapeutic intervention in a single test.

– Prof Maritha Kotze - Division of Anatomical Pathology at Stellenbosch University’s Faculty of Medicine and Health Sciences

It is our mission to continue to provide cutting-edge sequencing services in the South African context, with the highest possible amount and quality analytical data, in the shortest possible time-frame.

Please note: graphs and images of this article can be seen in the CAF Annual Report - click here