Publications by authors named "Richard C Caswell"

8 Publications

  • Page 1 of 1

Missense substitutions at a conserved 14-3-3 binding site in HDAC4 cause a novel intellectual disability syndrome.

HGG Adv 2021 Jan 14;2(1):100015. Epub 2021 Jan 14.

Exeter Genomics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter EX2 5DW, UK.

Histone deacetylases play crucial roles in the regulation of chromatin structure and gene expression in the eukaryotic cell, and disruption of their activity causes a wide range of developmental disorders in humans. Loss-of-function alleles of , a founding member of the class IIa deacetylases, have been reported in brachydactyly-mental retardation syndrome (BDMR). However, while disruption of HDAC4 activity and deregulation of its downstream targets may contribute to the BDMR phenotype, loss of HDAC4 function usually occurs as part of larger deletions of chromosome 2q37; BDMR is also known as chromosome 2q37 deletion syndrome, and the precise role of HDAC4 within the phenotype remains uncertain. Thus, identification of missense variants should shed new light on the role of HDAC4 in normal development. Here, we report seven unrelated individuals with a phenotype distinct from that of BDMR, all of whom have heterozygous missense variants that affect a major regulatory site of HDAC4, required for signal-dependent 14-3-3 binding and nucleocytoplasmic shuttling. Two individuals possess variants altering Thr244 or Glu247, whereas the remaining five all carry variants altering Pro248, a key residue for 14-3-3 binding. We propose that the variants in all seven individuals impair 14-3-3 binding (as confirmed for the first two variants by immunoprecipitation assays), thereby identifying deregulation of HDAC4 as a pathological mechanism in a previously uncharacterized developmental disorder.
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http://dx.doi.org/10.1016/j.xhgg.2020.100015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841527PMC
January 2021

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia.

Genet Med 2021 05 20;23(5):881-887. Epub 2021 Jan 20.

Duke University Health System, Durham, NC, USA.

Purpose: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene.

Methods: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual.

Results: We identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy.

Conclusion: This collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.
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http://dx.doi.org/10.1038/s41436-020-01076-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107131PMC
May 2021

Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants.

Clin Chem 2020 07;66(7):958-965

Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK.

Background: Babies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4α (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.

Methods: Droplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies' genotypes were ascertained postnatally by Sanger sequencing.

Results: Droplet digital PCR assays for GCK or HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no false-positive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.

Conclusions: This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.
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http://dx.doi.org/10.1093/clinchem/hvaa104DOI Listing
July 2020

Using Structural Analysis to Assess the Impact of Missense Variants in MEN1.

J Endocr Soc 2019 Dec 27;3(12):2258-2275. Epub 2019 Sep 27.

Institute of Biomedical and Clinical Science, University of Exeter School of Medicine, Exeter, United Kingdom.

Despite the rapid expansion in recent years of databases reporting either benign or pathogenic genetic variations, the interpretation of novel missense variants remains challenging, particularly for clinical or genetic testing laboratories where functional analysis is often unfeasible. Previous studies have shown that thermodynamic analysis of protein structure can discriminate between groups of benign and pathogenic missense variants. However, although structures exist for many human disease‒associated proteins, such analysis remains largely unexploited in clinical laboratories. Here, we analyzed the predicted effect of 338 known missense variants on the structure of menin, the gene product. Results provided strong discrimination between pathogenic and benign variants, with a threshold of >4 kcal/mol for the predicted change in stability, providing a strong indicator of pathogenicity. Subsequent analysis of seven novel missense variants identified during clinical testing of patients with MEN1 showed that all seven were predicted to destabilize menin by >4 kcal/mol. We conclude that structural analysis provides a useful tool in understanding the effect of missense variants in and that integration of proteomic with genomic data could potentially contribute to the classification of novel variants in this disease.
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http://dx.doi.org/10.1210/js.2019-00260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6846327PMC
December 2019

A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia.

Hum Mol Genet 2019 11;28(21):3543-3551

Department of Molecular Genetics, Royal Devon & Exeter NHS Foundation Trust, Barrack Road, Exeter, EX2 5DW, UK.

We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.
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http://dx.doi.org/10.1093/hmg/ddz200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927460PMC
November 2019

Heterozygous mutations affecting the protein kinase domain of cause a syndromic form of developmental delay and intellectual disability.

J Med Genet 2018 01 11;55(1):28-38. Epub 2017 Oct 11.

Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS Trust, Nottingham, UK.

Introduction: Recent evidence has emerged linking mutations in to syndromic congenital heart disease. We present here genetic and phenotypic data pertaining to 16 individuals with mutations.

Methods: Patients were investigated by exome sequencing, having presented with developmental delay and additional features suggestive of a syndromic cause.

Results: Our cohort comprised 16 individuals aged 4-16 years. All had developmental delay, including six with autism spectrum disorder. Common findings included feeding difficulties (15/16), structural cardiac anomalies (9/16), seizures (4/16) and abnormalities of the corpus callosum (4/11 patients who had undergone MRI). All had craniofacial dysmorphism, with common features including short, upslanting palpebral fissures, hypertelorism or telecanthus, medial epicanthic folds, low-set, posteriorly rotated ears and a small mouth with thin upper lip vermilion. Fifteen patients had predicted missense mutations, including five identical p.(Asn842Ser) substitutions and two p.(Gly717Arg) substitutions. One patient had a canonical splice acceptor site variant (c.2898-1G>A). All mutations were located within the protein kinase domain of CDK13. The affected amino acids are highly conserved, and in silico analyses including comparative protein modelling predict that they will interfere with protein function. The location of the missense mutations in a key catalytic domain suggests that they are likely to cause loss of catalytic activity but retention of cyclin K binding, resulting in a dominant negative mode of action. Although the splice-site mutation was predicted to produce a stable internally deleted protein, this was not supported by expression studies in lymphoblastoid cells. A loss of function contribution to the underlying pathological mechanism therefore cannot be excluded, and the clinical significance of this variant remains uncertain.

Conclusions: These patients demonstrate that heterozygous, likely dominant negative mutations affecting the protein kinase domain of the gene result in a recognisable, syndromic form of intellectual disability, with or without congenital heart disease.
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http://dx.doi.org/10.1136/jmedgenet-2017-104620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749303PMC
January 2018

Pitfalls of haplotype phasing from amplicon-based long-read sequencing.

Sci Rep 2016 Feb 17;6:21746. Epub 2016 Feb 17.

University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.

The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.
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http://dx.doi.org/10.1038/srep21746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756330PMC
February 2016

The HNF4A R76W mutation causes atypical dominant Fanconi syndrome in addition to a β cell phenotype.

J Med Genet 2014 Mar 27;51(3):165-9. Epub 2013 Nov 27.

Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, Devon, UK.

Background: Mutation specific effects in monogenic disorders are rare. We describe atypical Fanconi syndrome caused by a specific heterozygous mutation in HNF4A. Heterozygous HNF4A mutations cause a beta cell phenotype of neonatal hyperinsulinism with macrosomia and young onset diabetes. Autosomal dominant idiopathic Fanconi syndrome (a renal proximal tubulopathy) is described but no genetic cause has been defined.

Methods And Results: We report six patients heterozygous for the p.R76W HNF4A mutation who have Fanconi syndrome and nephrocalcinosis in addition to neonatal hyperinsulinism and macrosomia. All six displayed a novel phenotype of proximal tubulopathy, characterised by generalised aminoaciduria, low molecular weight proteinuria, glycosuria, hyperphosphaturia and hypouricaemia, and additional features not seen in Fanconi syndrome: nephrocalcinosis, renal impairment, hypercalciuria with relative hypocalcaemia, and hypermagnesaemia. This was mutation specific, with the renal phenotype not being seen in patients with other HNF4A mutations. In silico modelling shows the R76 residue is directly involved in DNA binding and the R76W mutation reduces DNA binding affinity. The target(s) selectively affected by altered DNA binding of R76W that results in Fanconi syndrome is not known.

Conclusions: The HNF4A R76W mutation is an unusual example of a mutation specific phenotype, with autosomal dominant atypical Fanconi syndrome in addition to the established beta cell phenotype.
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http://dx.doi.org/10.1136/jmedgenet-2013-102066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932761PMC
March 2014