Targeted exome sequencing integrated with clinicopathological information reveals novel and rare mutations in atypical, suspected and unknown cases of Alport syndrome or proteinuria

We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations

Developing Standard Treatment Workflows—way to universal healthcare in India

Primary healthcare caters to nearly 70% of the population in India and provides treatment for approximately 80–90% of common conditions. To achieve universal health coverage (UHC), the Indian healthcare system is gearing up by initiating several schemes such as National Health Protection Scheme, Ayushman Bharat, Nutrition Supplementation Schemes, and Inderdhanush Schemes. The healthcare delivery system is facing challenges such as irrational use of medicines, over- and under-diagnosis, high out-of-pocket expenditure, lack of targeted attention to preventive and promotive health services, and poor referral mechanisms. Healthcare providers are unable to keep pace with the volume of growing new scientific evidence and rising healthcare costs as the literature is not published at the same pace. In addition, there is a lack of common standard treatment guidelines, workflows, and reference manuals from the Government of India. Indian Council of Medical Research in collaboration with the National Health Authority, Govt. of India, and the WHO India country office has developed Standard Treatment Workflows (STWs) with the objective to be utilized at various levels of healthcare starting from primary to tertiary level care. A systematic approach was adopted to formulate the STWs. An advisory committee was constituted for planning and oversight of the process. Specialty experts' group for each specialty comprised of clinicians working at government and private medical colleges and hospitals. The expert groups prioritized the topics through extensive literature searches and meeting with different stakeholders. Then, the contents of each STW were finalized in the form of single-pager infographics. These STWs were further reviewed by an editorial committee before publication. Presently, 125 STWs pertaining to 23 specialties have been developed. It needs to be ensured that STWs are implemented effectively at all levels and ensure quality healthcare at an affordable cost as part of UHC

Monocyte chemotactic protein (MCP3) promoter polymorphism is associated with atopic asthma in the Indian population

This article does not have an abstract

Effect of sonic agitation, manual dynamic agitation on removal of Enterococcus faecalis biofilm

Objectives: The aim of the study was to compare manual dynamic agitation with sonic agitation on removal of intra radicular Enterococcus faecalis (E. faecalis) biofilm. Material and Methods: Extracted mandibular premolars for orthodontic purpose were sectioned at cervical level and divided into three groups (n = 30). The root canals were instrumented using Protaper rotary instruments up to apical file F4. Roots were sterilized and E. faecalis bacteria were incubated within their root canal space for four weeks. Confirmation of biofilm was done using scanning electron microscopy and Gram staining. All groups were irrigated with side vented needle by using three percent sodium hypochlorite (NaOCl) for 60 seconds. Two experimental groups were agitated with manual dynamic agitation (with master gutta-percha cone) and sonic agitation (EndoActivator). Remaining bacteria were collected using sterile paper point, which were incubated inside brain-heart infusion (BHI) broth to check turbidity. The turbid broth was streaked on blood agar plate for colony counts. Result: Both experimental groups showed highly significant difference in their mean colony count when compared with control group; with P < 0.001. Conclusion: Passive sonic agitation with EndoActivator has proven to be the best irrigating system followed by manual dynamic agitation and conventional needle irrigation

Validation and evolutionary conservation of the identified mutations in the patient with VUR and proteinuria.

<p>(A) Sanger sequencing analysis confirms the novel <i>COL4A3</i> mutations, <i>COL4A3_G/A_G695R</i> and <i>COL4A3_T/C_L1474P</i>, the <i>SALL2_G/C_G792R</i> and the <i>MYH9_C/T_L46F</i> mutations in the patient 1184405. (B) Multi species alignment shows that both the <i>COL4A3</i> mutation reference amino acids <i>Col4A3_ 695_G</i> and <i>Col4A3_1474_L</i>, the <i>SALL2_G792R</i> and the <i>MYH9_L46F</i> are highly conserved among species. (C) Schematic representation of Human <i>COL4A3</i> gene with the discovered mutations in patient 1184405. The domains are: signal peptide domain (red), the N terminal 7S domain (blue), the central triple helix collagenous (COL) domain (green) and the carboxy-terminal non-collagenous (NC1) domain (yellow).</p

Results of immunostaining with antibodies against α1, α3, and α5 chains of type IV collagen in the AS/VUR patient.

<p>(A) ColIVα1 expression is strong and consistent in the basement membranes (BM) of the glomeruli and the tubules; typically it is absent in adult glomeruli. Images show absent α3IV (B) and weak α5IV (C) staining in the glomerular BMs. (D&E) Electron micrograph from this patient shows segmental glomerular basement membrane (GBM) lamellation and outpouching (D arrow) and irregular thickening (E arrow) of the GBM characteristic of AS. COL4α3 immunostaining in control shows the normal tubular and capillary loop distribution for comparison (F).</p

Clinical characteristics of patients that underwent validation of custom multindexed targeted exome sequencing of 19 genes.

<p>Participants in italics are relatives in which the same mutation was found.</p

Pedigree analysis of the index patient with proetinuria and VUR.

<p>(A) Pedigree of the family of the index patient (1184405, arrow) shows presence of <i>Col4A3_T/C_L1474P</i> variant in one unaffected brother (4302442) and nephew (4302444), but absent in the unaffected sister (4302445) and niece (4302443); while the other damaging variant <i>Col4A3_G/A_G695R</i> is present only in the patient. The <i>SALL2</i> deleterious mutation <i>SALL2_G/C_G792R</i> is present in the patient and one unaffected brother (4302442) while the <i>MYH9_C/T_L46F</i> mutation is present in all but one affected family member (4302445). (B) Sanger sequencing analysis confirms the status of the novel <i>COL4A3</i> mutations <i>Col4A3_G/A_G695R</i> and <i>Col4A3_C/T_L1474P</i>, and the <i>SALL2_G/C_G792R</i> and <i>MYH9_C/T_L46F</i> mutations in the relatives of patient 1184405. The <i>Col4A3_T/C_L1474P</i> heterozygous variant was present in one unaffected brother (4302442) and nephew (4302444), but absent the unaffected sister (4302445) and niece (430443). The mutation <i>Col4A3_G/A_G695R</i> was absent in all other family members. The <i>SALL2_G/C_G792R</i> mutation was present only in the unaffected brother 4302442 while the <i>MYH9_C/T_L46F mutation</i> was more common and was present in everyone except the unaffected sister 4302445.</p