Recommendations for laboratory workflow that better support centralised amalgamation of genomic variant data: findings from CanVIG-UK national molecular laboratory survey.

BACKGROUND: National and international amalgamation of genomic data offers opportunity for research and audit, including analyses enabling improved classification of variants of uncertain significance. Review of individual-level data from National Health Service (NHS) testing of cancer susceptibility genes (2002-2023) submitted to the National Disease Registration Service revealed heterogeneity across participating laboratories regarding (1) the structure, quality and completeness of submitted data, and (2) the ease with which that data could be assembled locally for submission. METHODS: In May 2023, we undertook a closed online survey of 51 clinical scientists who provided consensus responses representing all 17 of 17 NHS molecular genetic laboratories in England and Wales which undertake NHS diagnostic analyses of cancer susceptibility genes. The survey included 18 questions relating to 'next-generation sequencing workflow' (11), 'variant classification' (3) and 'phenotypical context' (4). RESULTS: Widely differing processes were reported for transfer of variant data into their local LIMS (Laboratory Information Management System), for the formatting in which the variants are stored in the LIMS and which classes of variants are retained in the local LIMS. Differing local provisions and workflow for variant classifications were also reported, including the resources provided and the mechanisms by which classifications are stored. CONCLUSION: The survey responses illustrate heterogeneous laboratory workflow for preparation of genomic variant data from local LIMS for centralised submission. Workflow is often labour-intensive and inefficient, involving multiple manual steps which introduce opportunities for error. These survey findings and adoption of the concomitant recommendations may support improvement in laboratory dataflows, better facilitating submission of data for central amalgamation.

Cone-beam computed tomography assessment of maxillary anterior alveolar bone remodelling in extraction and non-extraction orthodontic cases using stable extra-alveolar reference.

OBJECTIVE: To explore alveolar cortical positional change in response to tooth movement in extraction and non-extraction orthodontic cases, using cone-beam computed tomography (CBCT) and stable extra-alveolar references. MATERIALS AND METHODS: The pre-treatment (T1) and post-treatment (T2) CBCT scans of 25 extraction (EXT) and matched 25 non-extraction (Non-EXT) orthodontic cases were imported into Dolphin Imaging 3D, and oriented uniformly. Sagittal and axial CBCT cross-sections were traced using customized software-generated guides. The displacement of teeth and alveolar bone cortices were automatically measured using the palatal plane (PP) and the line perpendicular to PP and passing Sella as reference. Intra- and inter-group differences between T1 and T2 were analysed. Subjects were also superimposed three-dimensionally using Geomagic Control X for qualitative analysis of cortical remodelling. RESULTS: The EXT group showed incisor retraction, while the Non-EXT group exhibited statistically significant incisor anterior tipping (P < .05). In EXT, both the labial and palatal cortices are resorbed. Non-EXT showed labial cortex anterior modelling, and statistically significant palatal cortex resorption (P < .05). In both groups, statistically significant decrease in total and palatal alveolar widths, increase in labial widths, and palatal dehiscence were observed. Comparatively, EXT showed significantly more incisal total and palatal width decrease and palatal vertical bone loss. CONCLUSION: Labial cortical remodelling was shown to follow anterior tooth movement, but the palatal cortical response to incisor retraction and labial cortical remodelling in general remained inconclusive. Narrowing of the alveolar housing and palatal dehiscence were observed regardless of extraction following orthodontic treatment.

Expert consensus guidelines for the genetic diagnosis of Alport syndrome.

Recent expert guidelines recommend genetic testing for the diagnosis of Alport syndrome. Here, we describe current best practice and likely future developments. In individuals with suspected Alport syndrome, all three COL4A5, COL4A3 and COL4A4 genes should be examined for pathogenic variants, probably by high throughput-targeted next generation sequencing (NGS) technologies, with a customised panel for simultaneous testing of the three Alport genes. These techniques identify up to 95% of pathogenic COL4A variants. Where causative pathogenic variants cannot be demonstrated, the DNA should be examined for deletions or insertions by re-examining the NGS sequencing data or with multiplex ligation-dependent probe amplification (MLPA). These techniques identify a further 5% of variants, and the remaining few changes include deep intronic splicing variants or cases of somatic mosaicism. Where no pathogenic variants are found, the basis for the clinical diagnosis should be reviewed. Genes in which mutations produce similar clinical features to Alport syndrome (resulting in focal and segmental glomerulosclerosis, complement pathway disorders, MYH9-related disorders, etc.) should be examined. NGS approaches have identified novel combinations of pathogenic variants in Alport syndrome. Two variants, with one in COL4A3 and another in COL4A4, produce a more severe phenotype than an uncomplicated heterozygous change. NGS may also identify further coincidental pathogenic variants in genes for podocyte-expressed proteins that also modify the phenotype. Our understanding of the genetics of Alport syndrome is evolving rapidly, and both genetic and non-genetic factors are likely to contribute to the observed phenotypic variability.

Four drug-sensitive subunits are required for maximal effect of a voltage sensor-targeted KCNQ opener.

KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers.

One drug-sensitive subunit is sufficient for a near-maximal retigabine effect in KCNQ channels.

Retigabine is an antiepileptic drug and the first voltage-gated potassium (Kv) channel opener to be approved for human therapeutic use. Retigabine is thought to interact with a conserved Trp side chain in the pore of KCNQ2-5 (Kv7.2-7.5) channels, causing a pronounced hyperpolarizing shift in the voltage dependence of activation. In this study, we investigate the functional stoichiometry of retigabine actions by manipulating the number of retigabine-sensitive subunits in concatenated KCNQ3 channel tetramers. We demonstrate that intermediate retigabine concentrations cause channels to exhibit biphasic conductance-voltage relationships rather than progressive concentration-dependent shifts. This suggests that retigabine can exert its effects in a nearly "all-or-none" manner, such that channels exhibit either fully shifted or unshifted behavior. Supporting this notion, concatenated channels containing only a single retigabine-sensitive subunit exhibit a nearly maximal retigabine effect. Also, rapid solution exchange experiments reveal delayed kinetics during channel closure, as retigabine dissociates from channels with multiple drug-sensitive subunits. Collectively, these data suggest that a single retigabine-sensitive subunit can generate a large shift of the KCNQ3 conductance-voltage relationship. In a companion study (Wang et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812014), we contrast these findings with the stoichiometry of a voltage sensor-targeted KCNQ channel opener (ICA-069673), which requires four drug-sensitive subunits for maximal effect.

Mobility shift of beta-dystroglycan as a marker of GMPPB gene-related muscular dystrophy.

BACKGROUND: Defects in glycosylation of alpha-dystroglycan (α-DG) cause autosomal-recessive disorders with wide clinical and genetic heterogeneity, with phenotypes ranging from congenital muscular dystrophies to milder limb girdle muscular dystrophies. Patients show variable reduction of immunoreactivity to antibodies specific for glycoepitopes of α-DG on a muscle biopsy. Recessive mutations in 18 genes, including guanosine diphosphate mannose pyrophosphorylase B (GMPPB), have been reported to date. With no specific clinical and pathological handles, diagnosis requires parallel or sequential analysis of all known genes. METHODS: We describe clinical, genetic and biochemical findings of 21 patients with GMPPB-associated dystroglycanopathy. RESULTS: We report eight novel mutations and further expand current knowledge on clinical and muscle MRI features of this condition. In addition, we report a consistent shift in the mobility of beta-dystroglycan (ß-DG) on Western blot analysis of all patients analysed by this mean. This was only observed in patients with GMPPB in our large dystroglycanopathy cohort. We further demonstrate that this mobility shift in patients with GMPPB was due to abnormal N-linked glycosylation of ß-DG. CONCLUSIONS: Our data demonstrate that a change in ß-DG electrophoretic mobility in patients with dystroglycanopathy is a distinctive marker of the molecular defect in GMPPB.

Congenital muscular dystrophies in the UK population: Clinical and molecular spectrum of a large cohort diagnosed over a 12-year period.

Congenital muscular dystrophies (CMDs) are clinically and genetically heterogeneous conditions; some fatal in the first few years of life and with central nervous system involvement, whereas others present a milder course. We provide a comprehensive report of the relative frequency and clinical and genetic spectrum of CMD in the UK. Genetic analysis of CMD genes in the UK is centralised in London and Newcastle. Between 2001 and 2013, a genetically confirmed diagnosis of CMD was obtained for 249 unrelated individuals referred to these services. The most common CMD subtype was laminin-α2 related CMD (also known as MDC1A, 37.4%), followed by dystroglycanopathies (26.5%), Ullrich-CMD (15.7%), SEPN1 (11.65%) and LMNA (8.8%) gene related CMDs. The most common dystroglycanopathy phenotype was muscle-eye-brain-like disease. Fifteen patients carried mutations in the recently discovered ISPD, GMPPB and B3GALNT2 genes. Pathogenic allelic mutations in one of the CMD genes were also found in 169 unrelated patients with milder phenotypes, such as limb girdle muscular dystrophy and Bethlem myopathy. In all, we identified 362 mutations, 160 of which were novel. Our results provide one of the most comprehensive reports on genetics and clinical features of CMD subtypes and should help diagnosis and counselling of families with this group of conditions.

Atomic basis for therapeutic activation of neuronal potassium channels.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

Congenital myopathies: Natural history of a large pediatric cohort.

OBJECTIVE: To assess the natural history of congenital myopathies (CMs) due to different genotypes. METHODS: Retrospective cross-sectional study based on case-note review of 125 patients affected by CM, followed at a single pediatric neuromuscular center, between 1984 and 2012. RESULTS: Genetic characterization was achieved in 99 of 125 cases (79.2%), with RYR1 most frequently implicated (44/125). Neonatal/infantile onset was observed in 76%. At birth, 30.4% required respiratory support, and 25.2% nasogastric feeding. Twelve percent died, mainly within the first year, associated with mutations in ACTA1, MTM1, or KLHL40. All RYR1-mutated cases survived and did not require long-term ventilator support including those with severe neonatal onset; however, recessive cases were more likely to require gastrostomy insertion (p = 0.0028) compared with dominant cases. Independent ambulation was achieved in 74.1% of all patients; 62.9% were late walkers. Among ambulant patients, 9% eventually became wheelchair-dependent. Scoliosis of variable severity was reported in 40%, with 1/3 of (both ambulant and nonambulant) patients requiring surgery. Bulbar involvement was present in 46.4% and required gastrostomy placement in 28.8% (at a mean age of 2.7 years). Respiratory impairment of variable severity was a feature in 64.1%; approximately half of these patients required nocturnal noninvasive ventilation due to respiratory failure (at a mean age of 8.5 years). CONCLUSIONS: We describe the long-term outcome of a large cohort of patients with CMs. While overall course is stable, we demonstrate a wide clinical spectrum with motor deterioration in a subset of cases. Severity in the neonatal/infantile period is critical for survival, with clear genotype-phenotype correlations that may inform future counseling.