Clinical relevance of double heterozygosity revealed by next-generation sequencing of homologous recombination repair pathway genes in South African breast cancer patients.

PURPOSE: Genetically predisposed breast cancer (BC) patients represent a minor but clinically meaningful subgroup of the disease, with 25% of all cases associated with actionable variants in BRCA1/2. Diagnostic implementation of next-generation sequencing (NGS) resulted in the rare identification of BC patients with double heterozygosity for deleterious variants in genes partaking in homologous recombination repair of DNA. As clinical heterogeneity poses challenges for genetic counseling, this study focused on the occurrence and clinical relevance of double heterozygous BC in South Africa. METHODS: DNA samples were diagnostically screened using the NGS-based Oncomine™ BRCA Expanded Research Assay. Data was generated on the Ion GeneStudio S5 system and analyzed using the Torrent Suite™ and reporter software. The clinical significance of the variants detected was determined using international variant classification guidelines and treatment implications. RESULTS: Six of 1600 BC patients (0.375%) tested were identified as being bi-allelic for two germline likely pathogenic or pathogenic variants. Most of the variants were present in BRCA1/2, including two founder-related small deletions in three cases, with family-specific variants detected in ATM, BARD1, FANCD2, NBN, and TP53. The scientific interpretation and clinical relevance were based on the clinical and tumor characteristics of each case. CONCLUSION: This study increased current knowledge of the risk implications associated with the co-occurrence of more than one pathogenic variant in the BC susceptibility genes, confirmed to be a rare condition in South Africa. Further molecular pathology-based studies are warranted to determine whether clinical decision-making is affected by the detection of a second pathogenic variant in BRCA1/2 and TP53 carriers.

Identification of a novel WAS mutation in a South African patient presenting with atypical Wiskott-Aldrich syndrome: a case report.

BACKGROUND: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in the WAS gene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes. CASE PRESENTATION: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identified c. 397 G > A as a novel hemizygous missense mutation located in exon 4 of WAS. CONCLUSION: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome.

Exome sequencing identifies a novel TTC37 mutation in the first reported case of Trichohepatoenteric syndrome (THE-S) in South Africa.

BACKGROUND: Trichohepatoenteric syndrome (THE-S) or phenotypic diarrhoea of infancy is a rare autosomal recessive disorder characterised by severe infantile diarrhoea, facial dysmorphism, immunodeficiency and woolly hair. It was first described in 1982 in two infants with intractable diarrhoea, liver cirrhosis and abnormal hair structure on microscopy. We report on two siblings from a consanguineous family of Somali descent who, despite extensive clinical investigation, remained undiagnosed until their demise. The index patient died of fulminant cytomegalovirus pneumonitis at 3 months of age. METHODS: Whole exome sequencing (WES) was performed on a premortem DNA sample from the index case. Variants in a homozygous recessive state or compound heterozygous state were prioritized as potential candidate variants using TAPER™. Sanger sequencing was done to genotype the parents, unaffected sibling and a deceased sibling for the variant of interest. RESULTS: Exome sequencing identified a novel homozygous mutation (c.4507C > T, rs200067423) in TTC37 which was confirmed by Sanger sequencing in the index case. The identification of this mutation led to the diagnosis of THE-S in the proband and the same homozygous variant was confirmed in a male sibling who died 4 years earlier with severe chronic diarrhoea of infancy. The unaffected parents and sister were heterozygous for the identified variant. CONCLUSIONS: WES permitted definitive genetic diagnosis despite an atypical presentation in the index case and suggests that severe infection, likely secondary to immunodeficiency, may be a presenting feature. In addition definitive molecular diagnosis allows for genetic counseling and future prenatal diagnosis, and demonstrates the value of WES for post-mortem diagnosis of disorders with a non-specific clinical presentation in which a Mendelian cause is suspected.

Genomic medicine and risk prediction across the disease spectrum.

Genomic medicine is based on the knowledge that virtually every medical condition, disease susceptibility or response to treatment is caused, regulated or influenced by genes. Genetic testing may therefore add value across the disease spectrum, ranging from single-gene disorders with a Mendelian inheritance pattern to complex multi-factorial diseases. The critical factors for genomic risk prediction are to determine: (1) where the genomic footprint of a particular susceptibility or dysfunction resides within this continuum, and (2) to what extent the genetic determinants are modified by environmental exposures. Regarding the small subset of highly penetrant monogenic disorders, a positive family history and early disease onset are mostly sufficient to determine the appropriateness of genetic testing in the index case and to inform pre-symptomatic diagnosis in at-risk family members. In more prevalent polygenic non-communicable diseases (NCDs), the use of appropriate eligibility criteria is required to ensure a balance between benefit and risk. An additional screening step may therefore be necessary to identify individuals most likely to benefit from genetic testing. This need provided the stimulus for the development of a pathology-supported genetic testing (PSGT) service as a new model for the translational implementation of genomic medicine in clinical practice. PSGT is linked to the establishment of a research database proven to be an invaluable resource for the validation of novel and previously described gene-disease associations replicated in the South African population for a broad range of NCDs associated with increased cardio-metabolic risk. The clinical importance of inquiry concerning family history in determining eligibility for personalized genotyping was supported beyond its current limited role in diagnosing or screening for monogenic subtypes of NCDs. With the recent introduction of advanced microarray-based breast cancer subtyping, genetic testing has extended beyond the genome of the host to also include tumor gene expression profiling for chemotherapy selection. The decreasing cost of next generation sequencing over recent years, together with improvement of both laboratory and computational protocols, enables the mapping of rare genetic disorders and discovery of shared genetic risk factors as novel therapeutic targets across diagnostic boundaries. This article reviews the challenges, successes, increasing inter-disciplinary integration and evolving strategies for extending PSGT towards exome and whole genome sequencing (WGS) within a dynamic framework. Specific points of overlap are highlighted between the application of PSGT and exome or WGS, as the next logical step in genetically uncharacterized patients for whom a particular disease pattern and/or therapeutic failure are not adequately accounted for during the PSGT pre-screen. Discrepancies between different next generation sequencing platforms and low concordance among variant-calling pipelines caution against offering exome or WGS as a stand-alone diagnostic approach. The public reference human genome sequence (hg19) contains minor alleles at more than 1 million loci and variant calling using an advanced major allele reference genome sequence is crucial to ensure data integrity. Understanding that genomic risk prediction is not deterministic but rather probabilistic provides the opportunity for disease prevention and targeted treatment in a way that is unique to each individual patient.

Clinical Utility of Whole Exome Sequencing and Targeted Panels for the Identification of Inborn Errors of Immunity in a Resource-Constrained Setting.

Primary immunodeficiency disorders (PIDs) are inborn errors of immunity (IEI) that cause immune system impairment. To date, more than 400 single-gene IEI have been well defined. The advent of next generation sequencing (NGS) technologies has improved clinical diagnosis and allowed for discovery of novel genes and variants associated with IEI. Molecular diagnosis provides clear clinical benefits for patients by altering management, enabling access to certain treatments and facilitates genetic counselling. Here we report on an 8-year experience using two different NGS technologies, namely research-based WES and targeted gene panels, in patients with suspected IEI in the South African healthcare system. A total of 52 patients' had WES only, 26 had a targeted gene panel only, and 2 had both panel and WES. Overall, a molecular diagnosis was achieved in 30% (24/80) of patients. Clinical management was significantly altered in 67% of patients following molecular results. All 24 families with a molecular diagnosis received more accurate genetic counselling and family cascade testing. Results highlight the clinical value of expanded genetic testing in IEI and its relevance to understanding the genetic and clinical spectrum of the IEI-related disorders in Africa. Detection rates under 40% illustrate the complexity and heterogeneity of these disorders, especially in an African population, thus highlighting the need for expanded genomic testing and research to further elucidate this.

Exome Sequencing Identifies a Novel MAP3K14 Mutation in Recessive Atypical Combined Immunodeficiency.

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met- versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches.

Implementation of a breast cancer genetic service in South Africa - lessons learned.

BACKGROUND: Genetic testing for BRCA mutations has been available in the Western Cape of South Africa since 2005, but practical implementation of genetic counselling and testing has been challenging. OBJECTIVE: To describe an approach to breast cancer genetic counselling and testing developed in a resource-constrained environment at Tygerberg Hospital in Cape Town, Western Cape. METHODS: Genetic counselling is offered in a stepwise manner to our diverse patient population, with a focus on affected probands, and subsequent cascade testing. A record review of BRCA testing between 2005 and 2011 was performed. RESULTS; During this period 302 probands received genetic testing, with increasing numbers tested over time. Of 1 520 women treated for breast cancer since 2008, 226 (14.9%) accepted BRCA testing, and 39 tested positive (17.3% of those tested, and 2.6% of all women). Common founder mutations were detected in 11.9% of women (36/302), and comprised 73% (36/49) of mutations detected. Cascade testing increased after 2010: 16 female and 4 male family members of 19 probands accepted testing, with 6 positives being detected. CONCLUSION: A protocol-driven approach focusing on probands, with initial pre-test counselling by primary care staff has proven effective in establishing the service. Involvement of a clinical geneticist/genetic counsellor has permitted more detailed post-test counselling and increased use of cascade testing.