Duchenne/becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner? in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.     

Detection and Characterization of a De Novo Alu Retrotransposition Event Causing NKX2-1-Related Disorder

Background

Heterozygous NKX2-1 loss-of-function variants cause combinations of hyperkinetic movement disorders (MDs, particularly childhood-onset chorea), pulmonary dysfunction, and hypothyroidism. Mobile element insertions (MEIs) are potential disease-causing structural variants whose detection in routine diagnostics remains challenging.

Objective

To establish the molecular diagnosis of two first-degree relatives with clinically suspected NKX2-1-related disorder who had negative NKX2-1 Sanger (SS), whole-exome (WES), and whole-genome (WGS) sequencing.

Methods

The proband's WES was analyzed for MEIs. A candidate MEI in NKX2-1 underwent optimized SS after plasmid cloning. Functional studies exploring NKX2-1 haploinsufficiency at RNA and protein levels were performed.

Results

A 347-bp AluYa5 insertion with a 65-bp poly-A tail followed by a 16-bp duplication of the pre-insertion wild-type sequence in exon 3 of NKX2-1 (ENST00000354822.7:c.556_557insAlu541_556dup) segregated with the disease phenotype.

Conclusions

We identified a de novo exonic AluYa5 insertion causing NKX2-1-related disorder in SS/WES/WGS-negative cases, suggesting that MEI analysis of short-read sequencing data or targeted long-read sequencing could unmask the molecular diagnosis of unsolved MD cases. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.     

The usefulness of electrodiagnostic studies in the diagnosis and management of neuromuscular disorders

Introduction: This study seeks to evaluate the usefulness of electrodiagnostic (EDX) studies in terms of the patient's diagnosis and subsequent management and to identify patient groups in which EDX is particularly useful. Methods: The records of new patients referred to a single tertiary hospital EDX laboratory during 1 calendar year were reviewed to determine whether results of EDX studies led to a changed diagnosis and/or management plan. Logistic regression was used to determine whether any factors were associated with changed diagnosis or management. Results: Results of EDX studies led to a change in diagnosis and a confirmation in diagnosis in 51.5% and 46.5% of the cases, respectively. Results of EDX studies led to a change in the management plan in 63.4% of all cases. The diagnosis and management plan were more likely to be changed in older patients and patients referred in hospital. Discussion: EDX studies seem useful for confirming or changing the diagnosis and in guiding management in patients with suspected neuromuscular disorders. Muscle Nerve 58 : 191–196, 2018     

Comparison of unilateral and bilateral complex finger tapping-related activation in premotor and primary motor cortex

Sixteen healthy right-handed subjects performed a complex finger-tapping task that broadly activates the motor and premotor regions, including primary motor (M1), ventral premotor (PMv), and dorsal premotor (PMd) cortex. This task was performed with the right hand only, left hand only and both hands simultaneously. Behavioral performance and the possibility of mirror movements were controlled through the use of MRI-compatible gloves to monitor finger movements. Using spatially normalized ROIs from the Human Motor Area Template (HMAT), comparisons were made of the spatial extent and location of activation in the left and right motor regions between all three tasks. During unilateral right and left hand tapping, ipsilateral precentral gyrus activation occurred in all subjects, mainly in the PMv and PMd. Ipsilateral M1 activation was less consistent and shifted anteriorly within M1, towards the border of M1 and premotor cortex. Regions of ipsilateral activation were also activated during contralateral and bilateral tasks. Overall, 83%/70%/58% of the ipsilaterally activated voxels in M1/PMd/PMv were also activated during contralateral and bilateral tapping. The mean percent signal change of spatially overlapping activated voxels was similar in PMv and PMd between all three tasks. However, the mean percent signal change of spatially overlapping M1 activation was significantly less during ipsilateral tapping compared with contra- or bilateral tapping. Results suggest that the ipsilateral fMRI activation in unilateral motor tasks may not be inhibitory in nature, but rather may reflect part of a bilateral network involved in the planning and/or execution of tapping in the ipsilateral hand.     

Chondrosarcoma of the bones of the feet

Twelve chondrosarcomas of the bones of the feet from 11 patients in the Scottish Bone Tumor Registry were reviewed. One patient with diaphyseal aclasis (osteochondromatosis) developed 2 chondrosarcomas. The mean age of patients was 52.3 years (range, 17 to 83 years). Men were predominantly affected. Four tumors affected the tarsal bones; the rest involved the short tubular bones. The usual clinical presentation was a painful, progressively enlarging swelling. Radiologically, most showed some bone expansion, cortical destruction with indistinct margins, and soft-tissue extension. Histologically, the majority were middle-grade tumors. Treatment included curettage or local excision for 4 tumors and amputation or ray resection for 8 tumors. Follow-up varied from 6 months to 18 years (average, 5.8 years). Local recurrence after surgery was seen in 3 patients. All 3 died because of metastases to the lungs or brain.     

Antibiotic prescribing for chronic skin wounds in primary care

The aim of this study was to describe and quantify systemic antibiotic prescribing for patients with chronic skin wounds presenting at the primary care, nonspecialist setting. Data for 1 year were extracted from a general practice morbidity database comprising approximately 185,000 patients attending family medical practitioners in Wales. Patients with chronic wounds (PCW) were identified using Read Codes and compared with nonwound patients who were randomly selected after matching for age-band, sex, and general practice. PCW received a significantly greater number of antibiotic courses than nonwound patients (p<0.001). This increased level of prescribing was evident for flucloxacillin, co-amoxiclav, cefaclor, cefalexin, erythromycin, trimethoprim, metronidazole, and ciprofloxacin (p<0.01 for all). While PCW also had a significantly higher prevalence of diabetes (16.5% compared with 6.6%, p<0.001), and attended at general practice significantly more frequently than nonwound patients (median (interquartile range) of 25 (17-40) visits per year compared with 12 (4-20), p<0.001), importantly, exclusion of diabetic patients and analysis of the proportion of visits on which patients received antibiotics did not affect the significance of the difference in antibiotic consumption. These data show a strong association between occurrence of chronic wounds and prescribing of antibiotics in primary health care, and wide variation in the type and duration of antibiotic therapy for chronic wounds. Further work is now indicated to rationalize this prescribing and determine the role that this exposure to antibiotics plays in the prevalence of antibiotic resistance in this at-risk elderly population.     

Accuracy of the national institute for neurological disorders and stroke/society for progressive supranuclear palsy and neuroprotection and natural history in Parkinson plus syndromes criteria for the diagnosis of progressive supranuclear palsy

Autopsy is the diagnostic gold standard for progressive supranuclear palsy (PSP). The National Institute of Neurological Disorders and Stroke and Society for Progressive Supranuclear Palsy (NINDS‐SPSP) criteria for the clinical diagnosis of “probable” PSP are thought to possess high specificity and low sensitivity. The NINDS‐SPSP criteria for “possible” PSP are considered to increase sensitivity at the expense of specificity. The Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) criteria are intended to improve sensitivity while maintaining high specificity. The aim of this study was to conduct a clinicopathological evaluation of the NINDS‐SPSP and NNIPPS criteria in tertiary neurological centers. Defined clinical features and their year of onset were recorded by chart review in neuropathologically diagnosed patients with PSP, Parkinsons's disease (PD), MSA parkinsonism and corticobasal degeneration from four European brain banks. Fulfilment of the clinical diagnostic criteria was verified for each year after disease onset and for the final antemortem record. We analyzed 98 PSP patients and 46 disease controls. The NINDS‐SPSP “probable” criteria yielded shorter time to diagnosis, slightly higher specificity and positive predictive value (PPV), and similar sensitivity, compared with the NNIPPS criteria. Unexpectedly, the NINDS‐SPSP “possible” criteria yielded the lowest sensitivity, specificity, and PPV. A combination of NINDS‐SPSP possible and probable criteria yielded the highest sensitivity. We suggest that the NINDS‐SPSP probable criteria might be preferred for recruitment of patients for clinical trials, where an early and specific diagnosis is important. For routine clinical care, where high sensitivity is crucial, a combination of NINDS possible and probable criteria might be preferred. © 2013 Movement Disorder Society     

Matrix metalloproteinase activity and immunohistochemical profile of matrix metalloproteinase-2 and -9 and tissue inhibitor of metalloproteinase-1 during human dermal wound healing

Proteolytic activity is required for the turnover of the extracellular matrix during wound healing. Matrix metalloproteinases can collectively cleave all components of the extracellular matrix, with the endogenous tissue inhibitor of metalloproteinase-1 regulating their activity. Breast tissue taken at varying postoperative times (n= 92) or during surgery (controls, n= 17), was used to investigate the temporal and spatial activity of matrix metalloproteinase-2 and -9 and tissue inhibitor of metalloproteinase-1 during human wound healing. Matrix metalloproteinase activity, determined using a quenched fluorescence substrate assay, increased during early healing (3-8 weeks) compared to controls, and then decreased between 24 and 36 weeks after surgery (p < 0.05 until 24 weeks, Mann-Whitney U-test). Immunohistochemistry scores for matrix metalloproteinase-9 expression were significantly elevated compared to controls in scar endothelial cells and fibroblasts from 2 until 12 and 20 weeks, respectively. Matrix metalloproteinase-2 staining was observed exclusively in fibroblasts, reaching maximum levels 8-12 weeks after surgery, decreasing by 1.5 years but remaining significantly increased. Tissue inhibitor of metalloproteinase-1 staining was relatively sparse but was significantly increased until 8 weeks after surgery. These results show that matrix metalloproteinases are present at elevated levels during early wound healing, when angiogenesis occurs, and suggest that matrix metalloproteinase-9 may play a significant role. The later expression of matrix metalloproteinase-2 and -9 in fibroblasts suggests a role in extracellular matrix remodeling.     

Effect of chronic antipsychotic treatment on striatal phosphodiesterase 10A levels: a [11C]MP-10 PET rodent imaging study with ex vivo confirmation

A number of phosphodiesterase 10A (PDE10) inhibitors are about to undergo clinical evaluation for their efficacy in treating schizophrenia. As phosphodiesterases are in the same signalling pathway as dopamine D₂ receptors, it is possible that prior antipsychotic treatment could influence these enzyme systems in patients. Chronic, in contrast to acute, antipsychotic treatment has been reported to increase brain PDE10A levels in rodents. The aim of this study was to confirm these findings in a manner that can be translated to human imaging studies to understand its consequences. Positron emission tomography (PET) scanning was used to evaluate PDE10A enzyme availability, after chronic haloperidol administration, using a specific PDE10A ligand ([¹¹C]MP-10). The binding of [¹¹C]MP-10 in the striatum and the cerebellum was measured in rodents and a simplified reference tissue model (SRTM) with cerebellum as the reference region was used to determine the binding potential (BP_ND). In rats treated chronically with haloperidol (2 mg kg⁻¹ per day), there was no significant difference in PDE10A levels compared with the vehicle-treated group (BP_ND±s.d.: 3.57±0.64 versus 2.86±0.71). Following PET scans, ex vivo analysis of striatal brain tissue for PDE10A mRNA (Pde10a) and PDE10A enzyme activity showed no significant difference. Similarly, the PDE10A protein content determined by western blot analysis was similar between the two groups, contrary to an earlier finding. The results of the study indicate that prior exposure to antipsychotic medication in rodents does not alter PDE10A levels.     

Psychophysiological correlates of dissociation, handedness, and hemispheric lateralization

Dissociation involves a disruption of typically integrated functions including consciousness, information perception, and memory; however, dissociation may not always be of a pathologic nature. Increasingly, studies are identifying relations between inconsistent handedness, mixed hemispheric lateralization, and dissociative symptomatology in both clinical and nonclinical populations. The current study explored whether a nonclinical sample of individuals scoring high in dissociation would display an inconsistent handedness in conjunction with a left hemispheric lateralization as measured by electroencephalography. Twenty-seven participants (12 males and 15 females) aged between 20 and 59 years (M = 29.1 year, SD = 11.2 years), completed the Dissociative Experiences Scale and Waterloo Handedness Questionnaire Revised after determining laterality. As predicted, inconsistently handed participants scoring high in dissociation displayed left hemispheric lateralization across frontal, central, and parietal regions. Conversely, right lateralization was found within Delta frequency band across temporal regions. The study provides a good framework for future research investigating the neurophysiological correlates underpinning dissociative symptomatology.