Eleanor G. Seaby

Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis

BACKGROUND Multiple genes underlying focal segmental glomerulosclerosis (FSGS) and/or steroid-resistant nephrotic syndrome (SRNS) have been identified, with the recent inclusion of collagen IV mutations responsible for Alport disease (AD) or thin basement membrane nephropathy (TBMN). We aimed to investigate the distribution of gene mutations in adult patients with primary FSGS/SRNS by targeted next generation sequencing (NGS). METHODS Eighty-one adults from 76 families were recruited; 24 families had a history of renal disease. A targeted NGS panel was designed and applied, covering 39 genes implicated in FSGS/SRNS including COL4A3-5. RESULTS Confirmed pathogenic mutations were found in 10 patients (6 with family history) from 9 families (diagnostic rate 12%). Probably pathogenic mutations were identified in an additional six patients (combined diagnostic rate 20%). Definitely pathogenic mutations were identified in 22% of patients with family history and 10% without. Mutations in COL4A3-5 were present in eight patients from six families, representing 56% of definitely pathogenic mutations, and establishing a diagnosis of AD in six patients and TBMN in two patients. Collagen mutations were identified in 38% of families with familial FSGS, and 3% with sporadic FSGS, with over half the mutations occurring in COL4A5. Patients with collagen mutations were younger at presentation and more likely to have family history, haematuria and glomerular basement membrane abnormalities. CONCLUSIONS We show that collagen IV mutations, including COL4A5, frequently underlie FSGS and should be considered, particularly with a positive family history. Targeted NGS improves diagnostic efficiency by investigating many candidate genes in parallel.

Deleterious coding variants in multi-case families with non-syndromic cleft lip and/or palate phenotypes

Nonsyndromic Cleft Lip and/or Palate (NSCLP) is regarded as a multifactorial condition in which clefting is an isolated phenotype, distinguished from the largely monogenic, syndromic forms which include clefts among a spectrum of phenotypes. Nonsyndromic clefting has been shown to arise through complex interactions between genetic and environmental factors. However, there is increasing evidence that the broad NSCLP classification may include a proportion of cases showing familial patterns of inheritance and contain highly penetrant deleterious variation in specific genes. Through exome sequencing of multi-case families ascertained in Bogota, Colombia, we identify 28 non-synonymous single nucleotide variants that are considered damaging by at least one predictive score. We discuss the functional impact of candidate variants identified. In one family we find a coding variant in the MSX1 gene which is predicted damaging by multiple scores. This variant is in exon 2, a highly conserved region of the gene. Previous sequencing has suggested that mutations in MSX1 may account for ~2% of NSCLP. Our analysis further supports evidence that a proportion of NSCLP cases arise through monogenic coding mutations, though further work is required to unravel the complex interplay of genetics and environment involved in facial clefting.

Mutations specific to the Rac-GEF domain of TRIO cause intellectual disability and microcephaly

Background Neurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1. Methods Whole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations. Results We identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation. Conclusions We describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.

Exome sequencing explained: a practical guide to its clinical application.

Next-generation sequencing has catapulted healthcare into a revolutionary genomics era. One such technology, whole-exome sequencing, which targets the protein-coding regions of the genome, has proven success in identifying new causal mutations for diseases of previously unknown etiology. With a successful diagnostic rate approaching 25% for rare disease in recent studies, its clinical utility is becoming increasingly popular. However, the interpretation of whole-exome sequencing data requires expertise in genomic informatics and clinical medicine to ensure the accurate and safe reporting of findings back to the bedside. This is challenged by vast amounts of sequencing data harbouring approximately 25 000 variants per sequenced individual. Computational strategies and fastidious filtering frameworks are thus required to extricate candidate variants in a sea of common polymorphisms. Once prioritized, identified variants require intensive scrutiny at a biological level, and require judicious assessment alongside the clinical phenotype. In the final step, all evidence is collated and documented alongside pathogenicity guidelines to produce an exome report that returns to the clinic. This review provides a practical guide for clinicians and genomic informaticians on the clinical application of whole-exome sequencing. We address sequencing capture and methodology, quality control parameters at different stages of sequencing analysis and propose an exome data filtering strategy that includes primary filtering (for the removal of probable benign variants) and secondary filtering for the prioritization of remaining candidates.

Can medical students accurately predict their learning? A study comparing perceived and actual performance in neuroanatomy.

It is important that clinicians are able to adequately assess their level of knowledge and competence in order to be safe practitioners of medicine. The medical literature contains numerous examples of poor self‐assessment accuracy amongst medical students over a range of subjects however this ability in neuroanatomy has yet to be observed. Second year medical students attending neuroanatomy revision sessions at the University of Southampton and the competitors of the National Undergraduate Neuroanatomy Competition were asked to rate their level of knowledge in neuroanatomy. The responses from the former group were compared to performance on a ten item multiple choice question examination and the latter group were compared to their performance within the competition. In both cohorts, self‐assessments of perceived level of knowledge correlated weakly to their performance in their respective objective knowledge assessments (r = 0.30 and r = 0.44). Within the NUNC, this correlation improved when students were instead asked to rate their performance on a specific examination within the competition (spotter, rS = 0.68; MCQ, rS = 0.58). Despite its inherent difficulty, medical student self‐assessment accuracy in neuroanatomy is comparable to other subjects within the medical curriculum. Anat Sci Educ 9: 488–495.

AMMECR1: a single point mutation causes developmental delay, midface hypoplasia and elliptocytosis

Background Deletions in the Xq22.3–Xq23 region, inclusive of COL4A5, have been associated with a contiguous gene deletion syndrome characterised by Alport syndrome with intellectual disability (Mental retardation), Midface hypoplasia and Elliptocytosis (AMME). The extrarenal biological and clinical significance of neighbouring genes to the Alport locus has been largely speculative. We sought to discover a genetic cause for two half-brothers presenting with nephrocalcinosis, early speech and language delay and midface hypoplasia with submucous cleft palate and bifid uvula. Methods Whole exome sequencing was undertaken on maternal half-siblings. In-house genomic analysis included extraction of all shared variants on the X chromosome in keeping with X-linked inheritance. Patient-specific mutants were transfected into three cell lines and microscopically visualised to assess the nuclear expression pattern of the mutant protein. Results In the affected half-brothers, we identified a hemizygous novel non-synonymous variant of unknown significance in AMMECR1 (c.G530A; p.G177D), a gene residing in the AMME disease locus. Transfected cell lines with the p.G177D mutation showed aberrant nuclear localisation patterns when compared with the wild type. Blood films revealed the presence of elliptocytes in the older brother. Conclusions Our study shows that a single missense mutation in AMMECR1 causes a phenotype of midface hypoplasia, mild intellectual disability and the presence of elliptocytes, previously reported as part of a contiguous gene deletion syndrome. Functional analysis confirms mutant-specific protein dysfunction. We conclude that AMMECR1 is a critical gene in the pathogenesis of AMME, causing midface hypoplasia and elliptocytosis and contributing to early speech and language delay, infantile hypotonia and hearing loss, and may play a role in dysmorphism, nephrocalcinosis and submucous cleft palate.

The benefits of being a near-peer teacher

Near‐peer teaching is used in anatomy education because of its benefits to the learner, teacher and faculty members. Despite the range of reports focusing on the learner, the advantages for the teacher, which are thought to include communication skills, subject knowledge and employability, are only beginning to be explored.

Commercial chicken breeds exhibit highly divergent patterns of linkage disequilibrium

The analysis of linkage disequilibrium (LD) underpins the development of effective genotyping technologies, trait mapping and understanding of biological mechanisms such as those driving recombination and the impact of selection. We apply the Malécot-Morton model of LD to create additive LD maps that describe the high-resolution LD landscape of commercial chickens. We investigated LD in chickens (Gallus gallus) at the highest resolution to date for broiler, white egg and brown egg layer commercial lines. There is minimal concordance between breeds of fine-scale LD patterns (correlation coefficient <0.21), and even between discrete broiler lines. Regions of LD breakdown, which may align with recombination hot spots, are enriched near CpG islands and transcription start sites (P<2.2 × 10−16), consistent with recent evidence described in finches, but concordance in hot spot locations between commercial breeds is only marginally greater than random. As in other birds, functional elements in the chicken genome are associated with recombination but, unlike evidence from other bird species, the LD landscape is not stable in the populations studied. The development of optimal genotyping panels for genome-led selection programmes will depend on careful analysis of the LD structure of each line of interest. Further study is required to fully elucidate the mechanisms underlying highly divergent LD patterns found in commercial chickens.

Thrombotic microangiopathy following haematopoietic stem cell transplant

Thrombotic microangiopathy is a potentially lethal complication of haematopoietic stem cell (bone marrow) transplantation. The pathophysiology is incompletely understood, although endothelial damage appears to be central. Platelet activation, neutrophil extracellular traps and complement activation appear to play key roles. Diagnosis may be difficult and universally accepted diagnostic criteria are not available. Treatment remains controversial. In some cases, withdrawal of calcineurin inhibitors is adequate. Rituximab and defibrotide also appear to have been used successfully. In severe cases, complement inhibitors such as eculizumab may play a valuable role. Further research is required to define the pathophysiology and determine both robust diagnostic criteria and the optimal treatment.

Inactivation of AMMECR1 is associated with growth, bone and heart alterations

We report five individuals with loss‐of‐function of the X‐linked AMMECR1: a girl with a balanced X‐autosome translocation and inactivation of the normal X‐chromosome; two boys with maternally inherited and de novo nonsense variants; and two half‐brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients’ features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patients cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid‐binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.