Maggie Williams

Exome Sequencing Identifies a DYNC1H1 Mutation in a Large Pedigree with Dominant Axonal Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease is characterized by length-dependent axonal degeneration with distal sensory loss and weakness, deep-tendon-reflex abnormalities, and skeletal deformities. It is caused by mutations in more than 40 genes. We investigated a four-generation family with 23 members affected by the axonal form (type 2), for which the common causes had been excluded by Sanger sequencing. Exome sequencing of three affected individuals separated by eight meioses identified a single shared novel heterozygous variant, c.917A>G, in DYNC1H1, which encodes the cytoplasmic dynein heavy chain 1 (here, novel refers to a variant that has not been seen in dbSNP131or the August 2010 release of the 1000 Genomes project). Testing of six additional affected family members showed cosegregation and a maximum LOD score of 3.6. The shared DYNC1H1 gene variant is a missense substitution, p.His306Arg, at a highly conserved residue within the homodimerization domain. Three mouse models with different mutations within this domain have previously been reported with age-related progressive loss of muscle bulk and locomotor ability. Cytoplasmic dynein is a large multisubunit motor protein complex and has a key role in retrograde axonal transport in neurons. Our results highlight the importance of dynein and retrograde axonal transport in neuronal function in humans.

EGFR and EGFRvIII analysis in glioblastoma as therapeutic biomarkers

Abstract Introduction. EGFR and EGFRvIII analysis is of current interest because of new EGFRvIII vaccine trials opened in the UK. EGFR activation promotes cellular proliferation via activation of MAPK and PI3K-Akt pathways. EGFRvIII is the most common variant resulting from an in-frame deletion of 801bp, leading to constitutively active EGFR. Method. 51 glioblastoma samples from a cohort of 50 patients were tested for EGFR amplification by FISH and immunohistochemistry and EGFRvIII expression by reverse-transcriptase PCR (RT-PCR), and immunohistochemistry. EGFR and EGFRvIII expression was compared with Overall Survival in the cohort. Results. Overall 22/51 samples (43%) were positive for EGFR, 16/51 (31%) were positive for EGFRvIII and 13/51 (25%) were positive for both. 9/51 cases (18%) were positive for EGFR alone, and 3/51 (6%) were positive for EGFRvIII alone. Of the EGFR positive cases, 22/51 (43%) were positive by FISH, 24/51 (47%) were positive by IHC and 2/51 (4%) were discrepant between methods (positive by IHC but non-amplified by FISH). Of the EGFRvIII positive cases, 16/51 (31%) were positive by RT-PCR, 17/51 (33%) were positive by IHC and 1/51 (2%) sample was discrepant (positive by IHC but not by RT-PCR). Neither EGFRvIII or EGFR are predictive of overall survival in this cohort. Conclusion. In our cohort, 25/51 (49%) of GBM showed EGFR alterations, including 16/51 (31%) with EGFRvIII. There was high concordance between IHC and FISH (96%) and IHC and RT-PCR (98%) as diagnostic methods. Neither EGFR or EGFRvIII is predictive of overall survival in this cohort. These results are key for selecting patients for novel individualised anti-EGFR therapies.

A comparison of methods for EGFR mutation testing in non-small cell lung cancer.

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non–small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

Barth syndrome without tetralinoleoyl cardiolipin deficiency: a possible ameliorated phenotype

Barth syndrome (BTHS) is an X-linked disorder characterised by cardiac and skeletal myopathy, growth delay, neutropenia and 3-methylglutaconic aciduria (3-MGCA). Patients have TAZ gene mutations which affect metabolism of cardiolipin, resulting in low tetralinoleoyl cardiolipin (CL4), an increase in its precursor, monolysocardiolipin (MLCL), and an increased MLCL/CL4 ratio. During development of a diagnostic service for BTHS, leukocyte CL4 was measured in 156 controls and 34 patients with genetically confirmed BTHS. A sub-group of seven subjects from three unrelated families was identified with leukocyte CL4 concentrations within the control range. This had led to initial false negative disease detection in two of these patients. MLCL/CL4 in this subgroup was lower than in other BTHS patients but higher than controls, with no overlap between the groups. TAZ gene mutations in these families are all predicted to be pathological. This report describes the clinical histories of these seven individuals with an atypical phenotype: some features were typical of BTHS (five have had cardiomyopathy, one family has a history of male infant deaths, three have growth delay and five have 3-MGCA) but none has persistent neutropenia, five have excellent exercise tolerance and two adults are asymptomatic. This report also emphasises the importance of measurement of MLCL/CL4 ratio rather than CL4 alone in the biochemical diagnosis of the BTHS.

Clinical and genetic aspects of KBG syndrome

KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by mutations in ANKRD11, one of the ankyrin repeat‐containing cofactors. We describe 32 KBG patients aged 2–47 years from 27 families ascertained via two pathways: targeted ANKRD11 sequencing (TS) in a group who had a clinical diagnosis of KBG and whole exome sequencing (ES) in a second group in whom the diagnosis was unknown. Speech delay and learning difficulties were almost universal and variable behavioral problems frequent. Macrodontia of permanent upper central incisors was seen in 85%. Other clinical features included short stature, conductive hearing loss, recurrent middle ear infection, palatal abnormalities, and feeding difficulties. We recognized a new feature of a wide anterior fontanelle with delayed closure in 22%. The subtle facial features of KBG syndrome were recognizable in half the patients. We identified 20 ANKRD11 mutations (18 novel: all truncating) confirmed by Sanger sequencing in 32 patients. Comparison of the two ascertainment groups demonstrated that facial/other typical features were more subtle in the ES group. There were no conclusive phenotype–genotype correlations. Our findings suggest that mutation of ANKRD11 is a common Mendelian cause of developmental delay. Affected patients may not show the characteristic KBG phenotype and the diagnosis is therefore easily missed. We propose updated diagnostic criteria/clinical recommendations for KBG syndrome and suggest that inclusion of ANKRD11 will increase the utility of gene panels designed to investigate developmental delay.

Chronic pain has a strong impact on quality of life in facioscapulohumeral muscular dystrophy

Earlier small case series and clinical observations reported on chronic pain playing an important role in facioscapulohumeral dystrophy (FSHD). The aim of this study was to determine the characteristics and impact of pain on quality of life (QoL) in patients with FSHD.

A unique triadin exon deletion causing a null phenotype

Large deletions and duplications may be responsible for some of the cause of genetically elusive arrhythmia syndromes. Introduction Triadin is a transmembrane protein located in the sarcoplasmic reticulum; it interacts with both ryanodine (RYR2) and calsequestrin (CASQ2) to facilitate calcium homeostasis in the human cardiac and skeletal muscle cells. Pathogenic variants in the RYR2 and CASQ2 genes are more commonly associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). Triadin is a more recently acknowledged protein, wherein genetic aberrations in triadin are responsible for a number of malignant arrhythmic syndromes, particularly in younger children. These triadin mutations are recessively inherited, and they are associated with a prolonged QT interval and T-wave abnormalities; the term “triadin knockout syndrome” has been suggested by Altmann and colleagues. The TRDN gene localizes to chromosome 6, and is seen in a variety of isoforms including Trisk 32, which is expressed predominantly in cardiac muscle. These isoforms are generated through alternative splicing of the triadin gene. The protein is composed of a transmembrane domain with both cytosolic and luminal components and is 286 amino acids long. Knockout of this gene in murine cardiac muscle causes a loss of calcium regulation, impaired excitation contraction coupling, and cardiac arrhythmia, particularly during betaadrenergic stimulation.

Clinical utility gene card for: Cantú syndrome

1.5 Mutational spectrum All reported variants to date are heterozygous single-nucleotide variants which are predicted to have an activating effect on the function of ATPsensitive potassium channels generated by the SUR2 and Kir6.1 proteins encoded by ABCC9 and KCNJ8. At least 15 different variants in ABCC9 and two variants in KCNJ8 have been reported to date1–4 (list of variants available in the GeneReviews entry for Cantú syndrome;5 see also the LOVD database for ABCC9 at http://databases.lovd.nl/shared/ genes/ABCC9). Variants in ABCC9 to date cluster in transmembrane domain 2, with ~80% of reported patients having variants in this domain. Recurrent ABCC9 variants affecting the amino acid residues p. Arg1116 and p.Arg1154 have been identified in approximately half of the individuals in whom pathogenic variants have been found to date. Copy number variants (small or large scale) have not been associated with Cantú syndrome.

A splice-site variant in ANKRD11 associated with classical KBG syndrome

To the editor: KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by pathogenic variants in ANKRD11, one of the ankyrin repeatcontaining cofactors. In our recent paper (Low et al., 2016) we described 32 patients with truncating variants in ANKRD11 and KBG syndrome. Goldenberg et al. (2016) simultaneously reported findings in 20 patients with KBG syndrome with truncating variants as well as a further 19 patients with microdeletions encompassing ANKRD11. The vast majority of pathogenic variants occur in exon 9 consistent with it containing >80% of the coding sequence. To our knowledge, there is only one previous report of a splicing variant in ANKRD11 associated with KBG syndrome. (Sirmaci et al. 2011) described a family in whom a c.7570-1G>C (p.Glu2524_Lys2525del) splice-acceptor site variant was present in all three affected individuals but absent in the unaffected mother. All three had classical features of KBG syndrome namely short stature, macrodontia, intellectual disability, delayed bone age, costovertebral anomalies, and hand anomalies as well as typical facial features. We report here a further case of KBG syndrome (Tekin et al., 2004) associatedwith a splice-site variant. The diagnosis wasmade at the age of 19 when the patient presented with intellectual disability and short stature (−2.25 SD). He was the only son of non-consanguineous parents. Only one parent was available for testing and did not have the variant. The other parent was not reported to have KBG syndrome related features. The pregnancy had been normal as had the birth, but parents described terrible feeding difficulties in infancy. He still does not like lumps in his food and has a poor appetite. There was marked speech delay with first words after 3 years old. However walking was normal at 12 months. He did not have any problems with otitis media and his hearing is normal. He has astigmatism. His bone age was delayed (8 years at a chronological age of 10 years). He has no known costovertebral anomalies but he has hand anomalies including brachydactyly and 5th finger clinodactyly. He has macrodontia of his secondary dentition and a very narrow palate. His health is otherwise generally good. His facial features are typical of KBG syndrome (see, Figure 1) with prominent bushy eyebrows, broad forehead with triangular shaped face, prominent cheekbones and long almond shaped palpebral fissures, a relatively thick nose with a rounded tip and a thin upper lip. The patient has intellectual disability and required extra help at school. He attends a college and is living independently with additional support. He has no significant behavioral issues but has “Asperger” traits and a formal diagnosis of ADHD. The c.7471-1G>C substitution that we detected via ANKRD11 sequence analysis is located in the acceptor splice site of intron 9 and is not present in the dbSNP, ExAC or the Exome Variant Server population datasets. In silico bioinformatic tools accessed via Alamut Visual v.2.7.1 (SplicesiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, and Human Splicing Finder) are concordant in predicting an effect on splicing, considered as very likely to cause a skip of exon 10 which encodes part of the D-box consensus sequence. This is supported by ANKRD11 reverse transcribed cDNA analysis from both the affected patient and his unaffected mother. A 298 bp band representing the WT ANKRD11 amplicon, consisting of exon 9 (partial)-exon 10exon 11 (partial), was clearly visible in both the affected patient and unaffected mother (Figure 2). An aberrant band of 199 bp was seen in the affected patient and absent from his mother (Figure 2). Sanger sequencing confirmed this to represent a transcript minus exon 10 (99 bp), supporting the in silico predications (Figure 3) of exon 10 skipping.

203 ELN Gene: UKGTN Service for SVAS and Cutis Laxa. Copy Number Variants (CNVS) Are a Common Cause of Disease

Pathogenic ELN gene mutations (ELN, MIM#130160) cause AD Supravalvular Aortic Stenosis (SVAS) a congenital narrowing of the ascending aorta, and Cutis Laxa (CL) characterised by inelastic, loose-hanging skin. Variable phenotype and penetrance is apparent. Pathogenic ELN variants result in loss of function and include frameshift (most common), nonsense, splice site and missense variants. The well characterised contiguous gene deletion syndrome, Williams-Beuren syndrome includes SVAS and encompasses at least 114kb on 7q11.23 including the ELN gene; however, there are only 5 case reports of CNVs within ELN (single or multiple exons). Bristol Genetics Laboratory provides a UKGTN approved service for ELN gene sequencing (33 coding exons). In three years, 52 UK and foreign patients with SVAS, CL or features such as pulmonary artery stenosis and aortic dilation have been tested. 18/52 (34%) patients were heterozygous for a likely pathogenic variant including frameshift (6), nonsense (4), splice (4), and missense (4). 12 of these cases were novel variants, 5 are supported by segregation analysis and 1 is sporadic. The remaining novel variants are classed as possibly pathogenic as they are phenotypically compatible. 12/35 patients negative on sequencing have so far been screened for CNVs by MLPA (MRC Holland) covering the Williams-Beuren syndrome region, including 10 exons of the ELN gene (1, 3, 4, 6, 9, 16, 20, 26, 27 and 33) and in addition a bespoke MLPA assay including probes for exons 28 to 30, 32 and 3’UTR. 4/12 (33%) patients have a heterozygous deletion within the ELNgene. A mother and daughter with pulmonary stenosis and an extended family history have a deletion spanning exons 30 to 33. This deletion was also identified in another patient with SVAS and arteriopathy. A deletion of the 5’ end of the gene, involving at least exon 1 (but not exon 3) was identified in an infant with SVAS and pulmonary branch stenosis, and a deletion involving the entire coding region of the ELN gene and at least the first two exons of the adjacent 3’ gene LIMK1 was detected in a neonate who died at 2 months with SVAS, pulmonary stenosis and mild hypoplasia with PDA. The deletion was detected in this patient’s father who consequentially was found to have an aortic regurgitation and in a subsequent pregnancy of this family which was lost at 31 weeks with pulmonary stenosis and significant aortic stenosis MLPA analysis has enhanced the clinical utility of this service giving an increased diagnostic yield in patients with SVAS and CL and related presentations.