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.

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.

Cystatin C and cystatin glomerular filtration rate as markers of early renal disease in Asian Indian subjects with glucose intolerance (CURES-32).

BACKGROUND: The aim of this study was to assess cystatin C (cys-C) as a marker of early diabetic nephropathy and cystatin glomerular filtration rate (cys-GFR) in Asian Indians. METHODS: Five groups of subjects were studied: Group 1, normal glucose tolerance (NGT) and normoalbuminuria (n = 43); group 2, impaired glucose tolerance (IGT) and normoalbuminuria (n = 44); group 3, type 2 diabetes (T2DM) with neither microalbuminuria nor retinopathy (n = 40); group 4, T2DM with microalbuminuria but without diabetic retinopathy (n = 40); and group 5, T2DM with microalbuminuria and any degree of diabetic retinopathy (DR) (n = 42). Subjects were recruited from the Chennai Urban Rural Epidemiology Study (CURES). Microalbumin concentration was assessed in the urine sample by immunoturbidometric assay. cys-C concentrations were measured in serum by a high-sensitivity particle-enhancing nephlometric assay. cys-GFR was calculated by the formula (86.7/cys-C) - 4.2. RESULTS: cys-C levels were highest in group 5 (1.75 +/- 0.12 mg/L) followed by group 4 (1.30 +/- 0.08 mg/L), group 3 (0.98 +/- 0.04 mg/L), group 2 (0.89 +/- 0.03 mg/L), and group 1 (0.79 +/- 0.18 mg/L, P < 0.001). cys-GFR levels were in reverse order going from highest in group 1, followed by groups 2, 3, 4, and 5. cys-C levels were correlated with age, fasting plasma glucose, glycosylated hemoglobin, microalbuminuria, and serum cholesterol. CONCLUSIONS: cys-C levels increase and cys-GFR levels decrease with increasing severity of glucose intolerance and are highest and lowest, respectively, in type 2 diabetes mellitus (T2DM) subjects with microalbuminuria and retinopathy. In T2DM subjects, cys-C and cys-GFR appear to be useful markers of early renal damage.