Profiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis

Background: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific ‘myocyte’ cultures. Methods From well-characterised MG patients we developed the ‘myocyte’ culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG). Results OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p  0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10− 6) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway. Conclusion Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies

A review of the genetic spectrum of hereditary spastic paraplegias, inherited neuropathies and spinal muscular atrophies in Africans

Background Genetic investigations of inherited neuromuscular disorders in Africans, have been neglected. We aimed to summarise the published data and comment on the genetic evidence related to inherited neuropathies (Charcot-Marie-Tooth disease (CMT)), hereditary spastic paraplegias (HSP) and spinal muscular atrophy (SMA) in Africans. Methods PubMed was searched for relevant articles and manual checking of references and review publications were performed for African-ancestry participants with relevant phenotypes and identified genetic variants. For each case report we extracted phenotype information, inheritance pattern, variant segregation and variant frequency in population controls (including up to date frequencies from the gnomAD database). Results For HSP, 23 reports were found spanning the years 2000–2019 of which 19 related to North Africans, with high consanguinity, and six included sub-Saharan Africans. For CMT, 19 reports spanning years 2002–2021, of which 16 related to North Africans and 3 to sub-Saharan Africans. Most genetic variants had not been previously reported. There were 12 reports spanning years 1999–2020 related to SMN1-SMA caused by homozygous exon 7 ± 8 deletion. Interestingly, the population frequency of heterozygous SMN1-exon 7 deletion mutations appeared 2 × lower in Africans compared to Europeans, in addition to differences in the architecture of the SMN2 locus which may impact SMN1-SMA prognosis. Conclusions Overall, genetic data on inherited neuromuscular diseases in sub-Saharan Africa, are sparse. If African patients with rare neuromuscular diseases are to benefit from the expansion in genomics capabilities and therapeutic advancements, then it is critical to document the mutational spectrum of inherited neuromuscular disease in Africa. Highlights Review of genetic variants reported in hereditary spastic paraplegia in Africans Review of genetic variants reported in genetic neuropathies in Africans Review of genetic underpinnings of spinal muscular atrophies in Africans Assessment of pathogenic evidence for candidate variant

Using Whole Genome Sequencing in an African Subphenotype of Myasthenia Gravis to Generate a Pathogenetic Hypothesis

Myasthenia gravis (MG) is a rare, treatable antibody-mediated disease which is characterized by muscle weakness. The pathogenic antibodies are most frequently directed at the acetylcholine receptors (AChRs) at the skeletal muscle endplate. An ophthalmoplegic subphenotype of MG (OP-MG), which is characterized by treatment resistant weakness of the extraocular muscles (EOMs), occurs in a proportion of myasthenics with juvenile symptom onset and African genetic ancestry. Since the pathogenetic mechanism(s) underlying OP-MG is unknown, the aim of this study was to use a hypothesis-generating genome-wide analysis to identify candidate OP-MG susceptibility genes and pathways. Whole genome sequencing (WGS) was performed on 25 AChR-antibody positive myasthenic individuals of African genetic ancestry sampled from the phenotypic extremes: 15 with OP-MG and 10 individuals with control MG (EOM treatment-responsive). Variants were called according to the Genome Analysis Toolkit (GATK) best practice guidelines using the hg38 reference genome. In addition to single variant association analysis, variants were mapped to genes (±200 kb) using VEGAS2 to calculate gene-based test statistics and HLA allele group assignment was inferred through “best-match” alignment of reads against the IMGT/HLA database. While there were no single variant associations that reached genome-wide significance in this exploratory sample, several genes with significant gene-based test statistics and known to be expressed in skeletal muscle had biological functions which converge on muscle atrophy signaling and myosin II function. The closely linked HLA-DPA1 and HLA-DPB1 genes were associated with OP-MG subjects (gene-based p &lt; 0.05) and the frequency of a functional A &gt; G SNP (rs9277534) in the HLA-DPB1 3′UTR, which increases HLA-DPB1 expression, differed between the two groups (G-allele 0.30 in OP-MG vs. 0.60 in control MG; p = 0.04). Furthermore, we show that rs9277534 is an HLA-DBP1 expression quantitative trait locus in patient-derived myocytes (p &lt; 1 × 10−3). The application of a SNP to gene to pathway approach to this exploratory WGS dataset of African myasthenic individuals, and comparing dichotomous subphenotypes, resulted in the identification of candidate genes and pathways that may contribute to OP-MG susceptibility. Overall, the hypotheses generated by this work remain to be verified by interrogating candidate gene and pathway expression in patient-derived extraocular muscle

Exome sequencing identifies novel dysferlin mutation in a family with paucisymptomatic heterozygous carriers

BACKGROUND: We investigated a South African family of admixed ancestry in which the first generation (G1) developed insidious progressive distal to proximal weakness in their twenties, while their offspring (G2) experienced severe unexpected symptoms of myalgia and cramps since adolescence. Our aim was to identify deleterious mutations that segregate with the affected individuals in this family. METHODS: Exome sequencing was performed on five cases, which included three affected G1 siblings and two pauci-symptomatic G2 offspring. As controls we included an unaffected G1 sibling and a spouse of one of the G1 affected individuals. Homozygous or potentially compound heterozygous variants that were predicted to be functional and segregated with the affected G1 siblings, were further evaluated. Additionally, we considered variants in all genes segregating exclusively with the affected (G1) and pauci-symptomatic (G2) individuals to address the possibility of a pseudo-autosomal dominant inheritance pattern in this family. RESULTS: All affected G1 individuals were homozygous for a novel truncating p.Tyr1433Ter DYSF (dysferlin) mutation, with their asymptomatic sibling and both pauci-symptomatic G2 offspring carrying only a single mutant allele. Sanger sequencing confirmed segregation of the variant. No additional potentially contributing variant was found in the DYSF or any other relevant gene in the pauci-symptomatic carriers. CONCLUSION: Our finding of a truncating dysferlin mutation confirmed dysferlinopathy in this family and we propose that the single mutant allele is the primary contributor to the neuromuscular symptoms seen in the second-generation pauci-symptomatic carriers

The Broad-Brush Survey Approach. A set of methods for rapid qualitative community assessment

Using a combination of qualitative data collection methods to collect data rapidly from a place on a particular topic is not a novel idea. Rapid participatory and qualitative appraisal approaches have been used in many different settings for the past 40 years, with the influential scholar Robert Chambers, and those he worked with, doing much to shape the practice from the 1970s. The methods spread beyond a rural, agriculture focus (Chambers, 1994) to embrace urban settings and the assessment of health and other areas of interest as well as settings in the Global North as well as South (Annett and Rifkin, 1995, Murray et al., 1994). I first used these approaches in the 1980s, while working in the Annapurna foothills in Nepal at an agricultural research station. We established the practice of a one week data collection exercise, which we called a `Combined Trek’ where a group of scientists from different disciplines, including me – a social anthropologist – systematically collected information using interviews, observations and discussions in a village and the surrounding area – working closely with the local people. Our purpose was to inform future agricultural interventions, building from what people were already doing. Cecilia Vindrola-Padros and Ginger Johnson (2020) detail in a review article how different qualitative methods have been adapted to be used to collect data rapidly. The need for speed, as they explain, has been a response to the increasing pressure many of us are under to deliver study findings quickly. Their review sets out how conventional methods have been adapted to be used rapidly in different settings. Among the combination of methods that they describe is the `rapid ethnographic assessment’. This assessment approach has grown as a response of anthropologists to pressure to produce results far more quickly that more conventional ethnographic approaches would allow. This set of methods is described in detail in the recent manual produced by Sangaramoorthy and Kroeger (2020). We are not, therefore, claiming that the approach set out in this manual is particularly novel nor indeed unique. The Broad Brush Survey, described in this manual is an approach originally developed by Valdo Pons (1993, 1996) and further developed and popularized through the work of Sandra Wallman (1996), which can be used to capture both the landscape and ‘feel’ of a community and the people in it. The research findings can be used to shape further investigators or interventions to address the problem at hand in a useful and practical manner rapidly, succinctly and systematically. This `Broad Brush Survey’ approach manual is, therefore, a contribution to the burgeoning literature on methods for rapid qualitative data collection methods and assessment. The use of the word ‘survey’ in the title of the set of methods may be perplexing to those who consider the term to be synonymous with `questionnaire’. This 6 is not the way we use the word – the Oxford English Dictionary offers several definitions of word `survey’, which include `the act of viewing, examining, or inspecting in detail [...] for some specific purpose’ and `the, or an, act of looking at something as a whole from a commanding position; a general or comprehensive look’. Both definitions convey the sense of our intention: to engage with, and in, a community for a short but concentrated period of time, seeking quickly, but thoroughly, to take a comprehensive look at the place for a specific purpose, and document the place at that moment in time. As we explain in the first chapter, the approach is systematic with a defined sequence of qualitative data collection methods, which gradually allows the user to build an understanding of place and people. The combination of methods used, however, is not set in stone and can be adapted to suit the purpose at hand. As such we hope that this manual serves as a guide to the possibilities which using this approach can offer both for those working in interdisciplinary projects as well as those from anthropology and sociology, for example, laying the groundwork for in-depth longitudinal research

Becoming a teacher

This book disseminates original research on learning in and from practice in pre-service teacher education. Authors such as Lederman and Lederman describe the student teaching practicum (or work-integrated learning [WIL]), which is an essential component of pre-service teacher education, as the ‘elephant in the room’. These authors note that 'the capstone experience in any teacher education programme is the student teaching practicum… [a]fter all, this is where the rubber hits the road'. However, many teacher educators will agree that this WIL component is sometimes very insufficient in assisting the student teacher to develop their own footing and voice as a teacher. This is the ‘gap’ that this research book addresses. Most of the chapters in the book report empirical data, with the exception of two chapters that can be categorized as systematic reviews. WIL is addressed from various angles in the chapters. Chapter 6 focuses on research related to what makes Finnish teacher education so effective, and in Chapter 4 researchers of the University of Johannesburg disseminate their findings on establishing a teaching school (based on Finnish insights) in Johannesburg. Chapter 3 highlights the challenges faced in open-and distance learning teacher education contexts. Several of the chapters disseminate research findings on alternative interventions to classic WIL, namely, where “safe spaces” or laboratories are created for student teachers to learn and grow professionally. These could either be simulations, such as software programmes and avatars in the intervention described in Chapter 2; student excursions, as the findings in chapters 5, 7 and 10 portray; or alternative approaches to WIL (e.g. Chapters 11 and 12). The book is devoted to scholarship in the field of pre-service teacher education. The target audience is scholars working in the fields of pre-service teacher education, work-integrated learning, and self-directed learning. The book makes a unique contribution in terms of firstly its extensive use of Cultural-Historical Activity Theory as a research lens, and secondly in drawing on various theoretical frameworks. Both quantitative and qualitative research informed the findings of the book

Increasing African genomic data generation and sharing to resolve rare and undiagnosed diseases in Africa: a call-to-action by the H3Africa rare diseases working group

The rich and diverse genomics of African populations is significantly underrepresented in reference and in disease-associated databases. This renders interpreting the Next Generation Sequencing (NGS) data and reaching a diagnostic more difficult in Africa and for the African diaspora. It increases chances for false positives with variants being misclassified as pathogenic due to their novelty or rarity. We can increase African genomic data by (1) making consent for sharing aggregate frequency data an essential component of research toolkit; (2) encouraging investigators with African data to share available data through public resources such as gnomAD, AVGD, ClinVar, DECIPHER and to use MatchMaker Exchange; (3) educating African research participants on the meaning and value of sharing aggregate frequency data; and (4) increasing funding to scale-up the production of African genomic data that will be more representative of the geographical and ethno-linguistic variation on the continent. The RDWG of H3Africa is hereby calling to action because this underrepresentation accentuates the health disparities. Applying the NGS to shorten the diagnostic odyssey or to guide therapeutic options for rare diseases will fully work for Africans only when public repositories include sufficient data from African subjects

Neuromuscular disease genetics in under-represented populations: increasing data diversity

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses ‘solved’ or ‘possibly solved’ ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% ‘solved’ and ∼13% ‘possibly solved’ outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally

Genetic variation in the immunosuppression pathway genes and breast cancer susceptibility : a pooled analysis of 42,510 cases and 40,577 controls from the Breast Cancer Association Consortium

Immunosuppression plays a pivotal role in assisting tumors to evade immune destruction and promoting tumor development. We hypothesized that genetic variation in the immunosuppression pathway genes may be implicated in breast cancer tumorigenesis. We included 42,510 female breast cancer cases and 40,577 controls of European ancestry from 37 studies in the Breast Cancer Association Consortium (2015) with available genotype data for 3595 single nucleotide polymorphisms (SNPs) in 133 candidate genes. Associations between genotyped SNPs and overall breast cancer risk, and secondarily according to estrogen receptor (ER) status, were assessed using multiple logistic regression models. Gene-level associations were assessed based on principal component analysis. Gene expression analyses were conducted using RNA sequencing level 3 data from The Cancer Genome Atlas for 989 breast tumor samples and 113 matched normal tissue samples. SNP rs1905339 (A > G) in the STAT3 region was associated with an increased breast cancer risk (per allele odds ratio 1.05, 95 % confidence interval 1.03-1.08; p value = 1.4 x 10(-6)). The association did not differ significantly by ER status. On the gene level, in addition to TGFBR2 and CCND1, IL5 and GM-CSF showed the strongest associations with overall breast cancer risk (p value = 1.0 x 10(-3) and 7.0 x 10(-3), respectively). Furthermore, STAT3 and IL5 but not GM-CSF were differentially expressed between breast tumor tissue and normal tissue (p value = 2.5 x 10(-3), 4.5 x 10(-4) and 0.63, respectively). Our data provide evidence that the immunosuppression pathway genes STAT3, IL5, and GM-CSF may be novel susceptibility loci for breast cancer in women of European ancestry.Peer reviewe