Targeted sequencing of the DMD locus: A comprehensive diagnostic tool for all mutations.

Background & objectives: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the. DMD: gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies. Methods: The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation. Results: In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene. Interpretation & conclusions: Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.

Efficacy of chimeric ectolysin P128 in drug-resistant Staphylococcus aureus bacteraemia in mice.

Objectives: P128 is a recombinant chimeric ectolysin with potent antistaphylococcal activity. P128 was evaluated as monotherapy and in combination with two standard-of-care (SoC) antibiotics, vancomycin and daptomycin, in mouse models of Staphylococcus aureus bacteraemia. Methods: Healthy BALB/c mice were challenged (intraperitoneally) with 109 cfu of MRSA strain COL or USA300 and treated with a single dose of P128 (0.2-10 mg/kg). Drug synergy was tested using a single dose of P128 (0.2 or 2.5 mg/kg) along with sub-therapeutic dose levels of vancomycin (27.5 or 55 mg/kg) or daptomycin (12.5 mg/kg). Bacterial load was checked in peritoneal fluid and in blood, at different time intervals. Synergy against drug-resistant strains was tested using the P128/vancomycin combination against vancomycin-resistant S. aureus (VRSA). Results: In MRSA bacteraemia, P128, vancomycin and daptomycin monotherapy resulted in 31%, 46% and 46% survival, respectively. The P128/vancomycin and P128/daptomycin combinations afforded increased survival of 85% and 88%, respectively. P128 showed a rapid bactericidal effect with a reduction of cfu in both the peritoneal fluid and the blood within 1 h. In VRSA bacteraemia, a mouse-equivalent therapeutic dose of vancomycin (110 mg/kg) failed to rescue animals. P128 (1-20 mg/kg) as monotherapy resulted in dose-dependent efficacy. Survival (37%) with 2.5 mg/kg P128 increased to 63% with the P128/vancomycin combination. Conclusions: P128 exerted a rapid bactericidal effect in vivo and rescued animals from fatal invasive MRSA and VRSA infections. P128/SoC antibiotic combinations exerted a synergistic effect. P128 restored the susceptibility of VRSA to vancomycin. P128 is a novel, potent therapeutic agent for antibiotic-resistant, systemic S. aureus infections.

Test-Retest Reliability of Tone Burst-Induced 500 Hz Air-Conduction Masseter Vestibular Evoked Myogenic Potential in Healthy Individuals.

BACKGROUND AND OBJECTIVES: Masseter vestibular evoked myogenic potential (mVEMP) is a newly developed tool which is used to assess the vestibulo-trigeminal neural and saccular functioning pathways. Recently, this test was added to a full test battery for evaluating the brainstem of people with neurological disorders and other vestibular diseases. For any test to qualify as a standard test, the test must have high reliability across all testing windows. Hence, the present study focused on validating the reliability of mVEMP in a large population. SUBJECTS AND METHODS: The study included 50 healthy participants with normal hearing. All the participants were tested using mVEMP and underwent retest within a month after the initial test. All parameters (latencies, peak-to-peak amplitude, asymmetric ratio) were observed for both sessions. To determine the statistically significant differences between and across the sessions, non-parametric tests such as Mann-Whitney U and Wilcoxon signed-rank tests were used. RESULTS: The test-retest reliability of all parameters were observed. The reliability was fair-to-good for P11 and N21 latencies. The other parameters showed less significance. There were no significant differences in sex and ear comparisons between and across the sessions. CONCLUSIONS: Our study demonstrated that the mVEMP is a reliable test which can be used to assess peripheral vestibular system dysfunction and neurological conditions.

Generation of two induced pluripotent stem cell (iPSC) lines from patients with Duchenne muscular dystrophy (IGIBi006-A and IGIBi008-A) carrying exonic deletions in the dystrophin gene.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with defect in dystrophin gene that shows features of degeneration of muscle tissue at an early age. Here, we describe iPSC lines generated from LCL of two patients of Indian origin carrying 46-48 and 49-50 exons deletions in DMD. The resulting iPSC lines IGIBi006-A and IGIBi008-A showed all the characteristic features of pluripotency, differentiated into cells of three germ layers in vitro and have no major genetic alterations due to reprogramming process. These lines can serve as a useful cell model for studying disease pathogenesis and will aid in precision therapy.

Matrilineal analysis of mutations in the DMD gene in a multigenerational South Indian cohort using DMD gene panel sequencing.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive irreversible muscle weakness, primarily of the skeletal and the cardiac muscles. DMD is characterised by mutations in the dystrophin gene, resulting in the absence or sparse quantities of dystrophin protein. A precise and timely molecular detection of DMD mutations encourages interventions such as carrier genetic counselling and in undertaking therapeutic measures for the DMD patients. RESULTS: In this study, we developed a 2.1 Mb custom DMD gene panel that spans the entire DMD gene, including the exons and introns. The panel also includes the probes against 80 additional genes known to be mutated in other muscular dystrophies. This custom DMD gene panel was used to identify single nucleotide variants (SNVs) and large deletions with precise breakpoints in 77 samples that included 24 DMD patients and their matrilineage across four generations. We used this panel to evaluate the inheritance pattern of DMD mutations in maternal subjects representing 24 DMD patients. CONCLUSION: Here we report our observations on the inheritance pattern of DMD gene mutations in matrilineage samples across four generations. Additionally, our data suggest that the DMD gene panel designed by us can be routinely used as a single genetic test to identify all DMD gene variants in DMD patients and the carrier mothers.

Sarcomatoid Carcinoma: "A Surgeons Nightmare".

Sarcomatoid carcinoma (SC) is a rare and unique disease of the oral cavity. It is a biphasic squamous cell carcinoma with sarcoma-like characteristics. It is a strikingly aggressive lesion with a rapid rate of growth and a high rate of metastasis. Diagnosing a sarcomatoid carcinoma may present a challenge; most can only be ascertained by immunohistochemical study. An aggressive treatment plan should be devised for this uncompromising disease with a readiness to accept the dire outcome. We hereby present a case of a 50-year-old male patient, with no history of deleterious habit, diagnosed with SC of the buccal mucosa that proved to be fatal.

'Sutureless' transconjunctival approach for infraorbital rim fractures.

AIM: To analyze the ease and surgical outcome of using sutureless transconjunctival approach for repair of infra-orbital fractures. DESIGN: Prospective clinical case series. MATERIALS AND METHODS: Totally 5 patients with infra-orbital rim or orbital floor fractures were selected and the fractures were accessed through a pre-septal transconjunctival incision. After reduction and fixation, the conjunctiva was just re-approximated and re-draped into position. Incidence of post-operative complications such as diplopia, lid retraction, eyelid dystopia, foreign body granuloma and poor conjunctival healing was assessed at intervals of 1 week, 15 days and a month post-operatively. RESULTS: No complications were observed in any of the 5 patients. Healing was satisfactory in all patients. CONCLUSION: The sutureless technique appears to be a time saving and technically simpler viable alternative to multilayered suturing in orbital trauma with minimal post-operative complications.

Frax prediction without BMD for assessment of osteoporotic fracture risk.

OBJECTIVE: To compare Fracture Risk Assessment Tool (FRAX) calculations with and without bone mineral density (BMD) in predicting the 10-year probability of hip and major osteoporotic fractures (MOF). METHODS: A cross-sectional review of patients requiring screening for osteoporosis as part of their routine medical care was conducted. Postmenopausal women and men over 50 years of age who were never diagnosed with osteoporosis or treated with U.S. Food and Drug Administration-approved agents for osteoporosis were included. Height, weight, FRAX questionnaire, femoral neck BMD, and T-score data were obtained. FRAX scores with BMD (FRAX/BMD) and without BMD (FRAX) were calculated. Subjects were separated on the basis of identical and different treatment recommendations. Fracture risk factors were compared between groups using simple Student's t test analysis of numerical variables and Fisher's exact test analysis of binary variables. RESULTS: Of 151 total subjects, 127 (84%) had identical fracture risk predictions with or without BMD included in the FRAX calculation. Thirty subjects met treatment criteria and 97 did not, but the FRAX prediction was the same with risk factors alone or with risk factors plus BMD. Age was the only risk factor that was significantly different between those with identical and different predictions (median age, 64.42 and 76.25 years, respectively; P<.001). CONCLUSION: In most cases, FRAX alone provides the same prediction as FRAX with BMD. Younger age is more indicative of an identical prediction.