Progressive myoclonic epilepsy with Fanconi syndrome

This report illustrates the difficulties in diagnosing complex cases and demonstrates how whole exome sequencing can resolve complex phenotypes

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.<br/

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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

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

8 páginasNonsyndromic 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

The benefits of being a near-peer teacher

Background: Near-peer teaching is used in anatomy education because of its benefits to the learner, teacher and faculty. 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. Method: A questionnaire was distributed to the teachers involved in anatomy near-peer teaching at the University of Southampton and Brighton and Sussex Medical School. This questionnaire was designed using 0-10 rating scales to assess teacher perspectives on their level of knowledge, teaching skills and enjoyment of teaching. Free text responses determined the teachers’ motivation and perceived benefits from the teaching. Results: Twenty-eight questionnaires were gathered (54.9% response rate) including 20 from Southampton and 8 from BSMS. Long term knowledge retention and better understanding of the material were rated 8.1 and 7.9 out of 10 respectively. Eight responses were from currently practising doctors, who rated how much they now use their teaching skills as doctors as 8.9 out of 10. Of the 8 doctors, 7 gained points for their foundation programme applications as a direct result of near-peer teaching. The most common motivator for engaging in teaching was to improve subject matter knowledge and the most common benefit was improved communication skills. Discussion: There are numerous advantages to being a near-peer teacher in medical school, which include knowledge improvement, transferrable professional skills and employability. These initial results support the hypothesised benefits to the teachers and provide a foundation for further longitudinal studies

Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Purpose: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. Methods: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. Results: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity. Conclusion: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.This research was funded by National Institute for Health Research (NIHR) and the NewLife Foundation. The Baralle lab is supported by NIHR Research Professorship to D.B. (RP-2016-07-011).published version, accepted version (6 month embargo), submitted versio

Impaired expression of metallothioneins contributes to allergen-induced inflammation in patients with atopic dermatitis

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis

Mutations specific to the Rac-GEF domain of <i>TRIO</i> 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.<br/

Identification of novel locus associated with coronary artery aneurysms and validation of loci for susceptibility to Kawasaki disease

Kawasaki disease (KD) is a paediatric vasculitis associated with coronary artery aneurysms (CAA). Genetic variants influencing susceptibility to KD have been previously identified, but no risk alleles have been validated that influence CAA formation. We conducted a genome-wide association study (GWAS) for CAA in KD patients of European descent with 200 cases and 276 controls. A second GWAS for susceptibility pooled KD cases with healthy paediatric controls from vaccine trials in the UK (n = 1609). Logistic regression mixed models were used for both GWASs. The susceptibility GWAS was meta-analysed with 400 KD cases and 6101 controls from a previous European GWAS, these results were further meta-analysed with Japanese GWASs at two putative loci. The CAA GWAS identified an intergenic region of chromosome 20q13 with multiple SNVs showing genome-wide significance. The risk allele of the most associated SNV (rs6017006) was present in 13% of cases and 4% of controls; in East Asian 1000 Genomes data, the allele was absent or rare. Susceptibility GWAS with meta-analysis with previously published European data identified two previously associated loci (ITPKC and FCGR2A). Further meta-analysis with Japanese GWAS summary data from the CASP3 and FAM167A genomic regions validated these loci in Europeans showing consistent effects of the top SNVs in both populations. We identified a novel locus for CAA in KD patients of European descent. The results suggest that different genes determine susceptibility to KD and development of CAA and future work should focus on the function of the intergenic region on chromosome 20q13