Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Induced Pluripotent Stem Cell Derived Human Lung Organoids to Map and Treat the SARS-CoV2 Infections In Vitro.

COVID-19 is the current day pandemic that has claimed around 1,054,604 lives globally till date. Moreover, the number of deaths is going to increase over the next few months until the pandemic comes to an end, and a second wave has also been reported in few countries. Most interestingly, the death rate among certain populations from the same COVID-19 infection is highly variable. For instance, the European populations show a very high death rate, in contrast to the populations from Chinese ethnicities. Amongst all the closed cases with an outcome (total recovered + total died), the death rate in Italy is 13%, Iran is 6%, China is 5%, Brazil is 3%, The United States of America is 2%, India 2%, Israel is 1% as of October 08, 2020. However, the percentage was higher during the early phase of the pandemic. Moreover, the global death rate amongst all the patients with an outcome is 4%. Here we have reviewed virus-transmitted various respiratory tract infections and postulated a better understanding of SARS-CoV2 using lung stem cell organoids in vitro. Hence, here we propose the strategies of understanding first the infectivity/severity ratio of COVID-19 infections using various ethnicity originated induced pluripotent stem cell-derived lung stem cell organoids in vitro. The greater the infectivity to severity ratio, the better the disease outcome with the value of 1 being the worst disease outcome. This strategy will be useful for understanding the infectivity/severity ratio of virus induced respiratory tract infections for a possible betterment of community-based disease management. Also, such a strategy will be useful for screening the effect of various antiviral drugs/repurposed drugs for their efficacy in vitro.

CD34 cells in somatic, regenerative and cancer stem cells: Developmental biology, cell therapy, and omics big data perspective.

The transmembrane phosphoglycoprotein protein CD34 has conventionally been regarded as a marker for hematopoietic progenitors. Its expression on these cells has been leveraged for cell therapy applications in various hematological disorders. More recently, the expression of CD34 has also been reported on cells of nonhematopoietic origin. The list includes somatic cells such as endothelial cells, fibrocytes and interstitial cells and regenerative stem cells such as corneal keratocytes, muscle satellite cells, and muscle-derived stem cells. Furthermore, its expression on some cancer stem cells (CSCs) has also been reported. Till date, the functional roles of this molecule have been implicated in a multitude of cellular processes including cell adhesion, signal transduction, and maintenance of progenitor phenotype. However, the complete understanding about this molecule including its developmental origins, its embryonic connection, and associated functions is far from complete. Here, we review our present understanding of the structure and putative functions of the CD34 molecule based upon our literature survey. We also probed various biological databases to retrieve data related to the expression and associated molecular functions of CD34. Such information, upon synthesis, is hence likely to provide the suitability of such cells for cell therapy. Moreover, we have also covered the existing cell therapy and speculated cell therapy applications of CD34+ cells isolated from various lineages. We have also attempted here to speculate the role(s) of CD34 on CSCs. Finally, we discuss number of large-scale proteomics and transcriptomics studies that have been performed using CD34+ cells.

Genetic analysis of the glucocerebrosidase gene in South Indian patients with Parkinson's disease.

BACKGROUND: Mutations in the glucocerebrosidase (GBA) gene have been associated with Parkinson's disease (PD). Several variants in the gene have been identified as risk factors for the development of PD, but there is difference in the prevalence of this mutation in various ethnic groups and countries. There is no published study related to this field on the Indian population. AIMS AND OBJECTIVES: The aim of the study was to investigate the frequency of mutations in the GBA gene in Indian patients with PD. MATERIALS AND METHODS: To perform the mutation analysis of the GBA gene, we amplified its entire coding region, spanning 11 exons and intron/exon junctions in three fragments, with a set of three primer pairs using the long polymerase chain reaction enzyme mix from Fermentas, Canada. RESULTS: We screened a total of 100 PD patients for mutations in the GBA gene. The sequence analysis identified the following five variants in this gene: IVS1 + 191G > C, IVS4 + 47G > A (rs. 2075569), IVS6 - 86A > G (rs. 114099990), IVS9 + 141A > G (rs. 28373017), and IVS10 + 3G > A. Of these, two variants IVS1 + 191G > C and IVS10 + 3G > A are novel, and the remaining three are known variants reported in the Single Nucleotide Polymorphism database (dbSNP). All the known variants were detected in homozygous as well as in heterozygous states. Both novel variants were identified in only one patient in a heterozygous state. CONCLUSION: GBA mutation may not be so common in Indian patients with PD as compared to the other ethnic populations. These findings need to be confirmed in larger studies.

Whole exome sequencing in an Indian family links Coats plus syndrome and dextrocardia with a homozygous novel CTC1 and a rare HES7 variation.

BACKGROUND: Coats plus syndrome is an autosomal recessive, pleiotropic, multisystem disorder characterized by retinal telangiectasia and exudates, intracranial calcification with leukoencephalopathy and brain cysts, osteopenia with predisposition to fractures, bone marrow suppression, gastrointestinal bleeding and portal hypertension. It is caused by compound heterozygous mutations in the CTC1 gene. CASE PRESENTATION: We encountered a case of an eight-year old boy from an Indian family with manifestations of Coats plus syndrome along with an unusual occurrence of dextrocardia and situs inversus. Targeted resequencing of the CTC1 gene as well as whole exome sequencing (WES) were conducted in this family to identify the causal variations. The identified candidate variations were screened in ethnicity matched healthy controls. The effect of CTC1 variation on telomere length was assessed using Southern blot. A novel homozygous missense mutation c.1451A > C (p.H484P) in exon 9 of the CTC1 gene and a rare 3'UTR known dbSNP variation (c.*556 T > C) in HES7 were identified as the plausible candidates associated with this complex phenotype of Coats plus and dextrocardia. This CTC1 variation was absent in the controls and we also observed a reduced telomere length in the affected individual's DNA, suggesting its likely pathogenic nature. The reported p.H484P mutation is located in the N-terminal 700 amino acid regionthat is important for the binding of CTC1 to ssDNA through its two OB domains. WES data also showed a rare homozygous missense variation in the TEK gene in the affected individual. Both HES7 and TEK are targets of the Notch signaling pathway. CONCLUSIONS: This is the first report of a genetically confirmed case of Coats plus syndrome from India. By means of WES, the genetic variations in this family with unique and rare complex phenotype could be traced effectively. We speculate the important role of Notch signaling in this complex phenotypic presentation of Coats plus syndrome and dextrocardia. The present finding will be useful for genetic diagnosis and carrier detection in the family and for other patients with similar disease manifestations.

Scaling up robust immunopeptidomics technologies for a global T cell surveillance digital network.

The human immunopeptidome plays a central role in disease susceptibility and resistance. In our opinion, the development of immunopeptidomics and other peptide sequencing technologies should be prioritized during the next decade, particularly within the framework of the Human Immunopeptidome Project initiative. In this context, we present bold ideas, fresh arguments, and call upon industrial partners and funding organizations to support and champion this important initiative that we believe has the potential to save countless lives in the future.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature.

PURPOSE: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. METHODS: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. RESULTS: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. CONCLUSIONS: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.

Development of multiple reaction monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock.

In the present study, a targeted multiple reaction monitoring-mass spectrometry (MRM-MS) approach was developed to screen and identify protein biomarkers for brucellosis in humans and livestock. The selection of proteotypic peptides was carried out by generating in silico tryptic peptides of the Brucella proteome. Using bioinformatics analysis, 30 synthetic peptides corresponding to 10 immunodominant Brucella abortus proteins were generated. MRM-MS assays for the accurate detection of these peptides were optimized using 117 serum samples of human and livestock stratified as clinically confirmed (45), suspected (62), and control (10). Using high throughput MRM assays, transitions for four peptides were identified in several clinically confirmed and suspected human and livestock serum samples. Of these, peptide NAIYDVVTR corresponding to B. abortus proteins: BruAb2_0537 was consistently detected in the clinically confirmed serum samples of both humans and livestock with 100% specificity. To conclude, a high throughput MRM-MS-based protocol for detecting endogenous B. abortus peptides in serum samples of humans and livestock was developed. The developed protocol will help design sensitive assays to accurately diagnose brucellosis in humans and livestock. The data associated with this study are deposited in Panorama Public (https://panoramaweb.org/rNOZCy.url with ProteomeXchange ID: PXD034407).

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy.

PURPOSE: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. METHODS: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. RESULTS: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. CONCLUSIONS: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Sca1+ Progenitor Cells (Ex vivo) Exhibits Differential Proteomic Signatures From the Culture Adapted Sca1+ Cells (In vitro), Both Isolated From Murine Skeletal Muscle Tissue.

Stem cell antigen-1 (Sca-1) is a glycosyl-phosphatidylinositol-anchored membrane protein that is expressed in a sub-population of muscle stem and progenitor cell types. Reportedly, Sca-1 regulates the myogenic property of myoblasts and Sca-1-/- mice exhibited defective muscle regeneration. Although the role of Sca-1 in muscle development and maintenance is well-acknowledged, molecular composition of muscle derived Sca-1+ cells is not characterized. Here, we applied a high-resolution mass spectrometry-based workflow to characterize the proteomic landscape of mouse hindlimb skeletal muscle derived Sca-1+ cells. Furthermore, we characterized the impact of the cellular microenvironments on the proteomes of Sca-1+ cells. The proteome component of freshly isolated Sca-1+ cells (ex vivo) was compared with that of Sca-1+ cells expanded in cell culture (in vitro). The analysis revealed significant differences in the protein abundances in the two conditions reflective of their functional variations. The identified proteins were enriched in various biological pathways. Notably, we identified proteins related to myotube differentiation, myotube cell development and myoblast fusion. We also identified a panel of cell surface marker proteins that can be leveraged in future to enrich Sca-1+ cells using combinatorial strategies. Comparative analysis implicated the activation of various pathways leading to increased protein synthesis under in vitro condition. We report here the most comprehensive proteome map of Sca-1+ cells that provides insights into the molecular networks operative in Sca-1+ cells. Importantly, through our work we generated the proteomic blueprint of protein abundances significantly altered in Sca-1+ cells under ex vivo and in vitro conditions. The curated data can also be visualized at https://yenepoya.res.in/database/Sca-1-Proteomics .

Predicted peptide patterns from the SARS-CoV-2 proteome for MS-MS based diagnosis.

COVID-19 caused by 2019 novel coronavirus (2019-nCoV2) also known as SARS-CoV-2 has manifested globally since January 2020. COVID-19 was declared as a pandemic by the WHO and has become a serious global health concern. Real-time PCR based and antibody-based assays are being used for the clinical detection of the virus in body fluids and nasopharyngeal swabs. Antibody variability linked to viral mutations is a big concern. Hence, it is of interest to use data patterns from mass spectrometry-based platforms for the identification of SARS-CoV-2. This dataset can be used to perform targeted mass-spectrometric analysis of SARS-CoV-2 peptides. This work can be extrapolated for the detection of SARS-CoV-2 viral peptides in complex biological fluids for early diagnosis of COVID-19.

Single-nucleotide polymorphisms of methylenetetrahydrofolate reductase gene in a South Indian cohort with nonsyndromic cleft lip with or without palate.

OBJECTIVE: Clefts of the lip, with or without cleft palate and cleft palate only, collectively called as orofacial clefts (OFCs) are one of the most common congenital malformations with varying degrees of penetrance and phenotype expressions. The aim of this study was to investigate the association between methylenetetrahydrofolate reductase (MTHFR) cytosine-to-thymine (c. 677 C>T), adenine-to-cytosine (c.1298 A>C) single- nucleotide polymorphisms (SNPs) and South Indian patients with the nonsyndromic cleft lip with or without palate (NSCL ± P). METHODS: A cohort consisting of 25 cases of NSCL ± P and 18 controls from a South Indian cohort were included in this case-control study. Genetic analysis of c.677C>T and c.1298A C polymorphisms in the MTHFR gene was carried out using Sanger sequencing and analyzed from chromatogram profiles. Data interpretation was done using statistical software MedCalc Statistical Software version 16.2 and the Statistical Package for the Social Sciences (SPSS version 22.0). RESULTS: DNA sequence analysis of the MTHFR gene revealed c. 677C>T and c. 1298A>C polymorphisms in 16% and 76% of NSCL ± P cases, respectively. Heterozygous variant in MTHFR c. 1298A>C polymorphism was found to be a significant risk factor (P = 0.0164) for NSCL ± P in South Indian ethnic population. c.677C>T polymorphism, in particular, was apparently dormant overall in the study population. These results offer certain novelty in terms of the distinctive pattern in SNPs of genotypes observed in the study. CONCLUSION: NSCL ± P is one of the most common and challenging congenital malformations with complex etiological basis. Common risk factors such as MTHFR SNPs, namely c.677C>T and c.1298A>C, are subjected to variations in terms of ethnic group, geographic region and micro/macro-environmental factors. Overall, our study has explored part of South Indian ethnic population and revealed a different and unique distribution of mutations in this sample population.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2',7'-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Novel lysophosphatidic acid receptor 6 antagonists inhibit hepatocellular carcinoma growth through affecting mitochondrial function.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and the commonest liver cancer. It is expected to become the third leading cause of cancer-related deaths in Western countries by 2030. Effective pharmacological approaches for HCC are still unavailable, and the currently approved systemic treatments are unsatisfactory in terms of therapeutic results, showing many side effects. Thus, searching for new effective and nontoxic molecules for HCC treatment is of paramount importance. We previously demonstrated that lysophosphatidic acid (LPA) is an important contributor to the pathogenesis of HCC and that lysophosphatidic acid receptor 6 (LPAR6) actively supports HCC tumorigenicity. Here, we screened for novel LPAR6 antagonists and found that two compounds, 4-methylene-2-octyl-5-oxotetra-hydrofuran-3-carboxylic acid (C75) and 9-xanthenylacetic acid (XAA), efficiently inhibit HCC growth, both in vitro and in vitro, without displaying toxic effects at the effective doses. We further investigated the mechanisms of action of C75 and XAA and found that these compounds determine a G1-phase cell cycle arrest, without inducing apoptosis at the effective doses. Moreover, we discovered that both molecules act on mitochondrial homeostasis, by increasing mitochondrial biogenesis and reducing mitochondrial membrane potential. Overall, our results show two newly identified LPAR6 antagonists with a concrete potential to be translated into effective and side effect-free molecules for HCC therapy.

Sodium fluoride induced skeletal muscle changes: Degradation of proteins and signaling mechanism.

Fluoride is a well-known compound for its usefulness in healing dental caries. Similarly, fluoride is also known for its toxicity to various tissues in animals and humans. It causes skeletal fluorosis leading to osteoporosis of the bones. We hypothesized that when bones are affected by fluoride, the skeletal muscles are also likely to be affected by underlying molecular events involving myogenic differentiation. Murine myoblasts C2C12 were cultured in differentiation media with or without NaF (1 ppm-5 ppm) for four days. The effects of NaF on myoblasts and myotubes when exposed to low (1.5 ppm) and high concentration (5 ppm) were assessed based on the proliferation, alteration in gene expression, ROS production, and production of inflammatory cytokines. Changes based on morphology, multinucleated myotube formation, expression of MyHC1 and signaling pathways were also investigated. Concentrations of NaF tested had no effects on cell viability. NaF at low concentration (1.5 ppm) caused myoblast proliferation and when subjected to myogenic differentiation it induced hypertrophy of the myotubes by activating the IGF-1/AKT pathway. NaF at higher concentration (5 ppm), significantly inhibited myotube formation, increased skeletal muscle catabolism, generated reactive oxygen species (ROS) and inflammatory cytokines (TNF-α and IL-6) in C2C12 cells. NaF also enhanced the production of muscle atrophy-related genes, myostatin, and atrogin-1. The data suggest that NaF at low concentration can be used as muscle enhancing factor (hypertrophy), and at higher concentration, it accelerates skeletal muscle atrophy by activating the ubiquitin-proteosome pathway.

Genetic analysis of ATP7B in 102 south Indian families with Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder, characterized by excessive deposition of copper in various parts of the body, mainly in the liver and brain. It is caused by mutations in ATP7B. We report here the genetic analysis of 102 WD families from a south Indian population. Thirty-six different ATP7B mutations, including 13 novel ones [p.Ala58fs*19, p.Lys74fs*9, p.Gln281*, p.Pro350fs*12, p.Ser481*, p.Leu735Arg, p.Val752Gly, p.Asn812fs*2, p.Val845Ala, p.His889Pro, p.Ile1184fs*1, p.Val1307Glu and p.Ala1339Pro], were identified in 76/102 families. Interestingly, the mutation analysis of affected individuals in two families identified two different homozygous mutations in each family, and thus each affected individual from these families harbored two mutations in each ATP7B allele. Of 36 mutations, 28 were missense, thus making them the most prevalent mutations identified in the present study. Nonsense, insertion and deletion represented 3/36, 2/36 and 3/36 mutations, respectively. The haplotype analysis suggested founder effects for all the 14 recurrent mutations. Our study thus expands the mutational landscape of ATP7B with a total number of 758 mutations. The mutations identified during the present study will facilitate carrier and pre-symptomatic detection, and prenatal genetic diagnosis in affected families.

Integrated Multi-Omic Analysis of Mycobacterium tuberculosis H37Ra Redefines Virulence Attributes.

H37Ra is a virulence attenuated strain of Mycobacterium tuberculosis widely employed as a model to investigate virulence mechanisms. Comparative high-throughput studies have earlier correlated its avirulence to the presence of specific mutations or absence of certain proteins. However, a recent sequencing study of H37Ra, has disproved several genomic differences earlier reported to be associated with virulence. This warrants further investigations on the H37Ra proteome as well. In this study, we carried out an integrated analysis of the genome, transcriptome, and proteome of H37Ra. In addition to confirming single nucleotide variations (SNVs) and insertion-deletions that were reported earlier, our study provides novel insights into the mutation spectrum in the promoter regions of 7 genes. We also provide transcriptional and proteomic evidence for 3,900 genes representing ~80% of the total predicted gene count including 408 proteins that have not been identified previously. We identified 9 genes whose coding potential was hitherto reported to be absent in H37Ra. These include 2 putative virulence factors belonging to ESAT-6 like family of proteins. Furthermore, proteogenomic analysis enabled us to identify 63 novel proteins coding genes and correct 25 existing gene models in H37Ra genome. A majority of these were found to be conserved in the virulent strain H37Rv as well as in other mycobacterial species suggesting that the differences in the virulent and avirulent strains of M. tuberculosis are not entirely dependent on the expression of certain proteins or their absence but may possibly be ascertained to functional changes.

Genetic Analysis of PLA2G6 in 22 Indian Families with Infantile Neuroaxonal Dystrophy, Atypical Late-Onset Neuroaxonal Dystrophy and Dystonia Parkinsonism Complex.

Mutations in PLA2G6 were identified in patients with a spectrum of neurodegenerative conditions, such as infantile neuroaxonal dystrophy (INAD), atypical late-onset neuroaxonal dystrophy (ANAD) and dystonia parkinsonism complex (DPC). However, there is no report on the genetic analysis of families with members affected with INAD, ANAD and DPC from India. Therefore, the main aim of this study was to perform genetic analysis of 22 Indian families with INAD, ANAD and DPC. DNA sequence analysis of the entire coding region of PLA2G6 identified 13 different mutations, including five novel ones (p.Leu224Pro, p.Asp283Asn, p.Arg329Cys, p.Leu491Phe, and p.Arg649His), in 12/22 (54.55%) families with INAD and ANAD. Interestingly, one patient with INAD was homozygous for two different mutations, p.Leu491Phe and p.Ala516Val, and thus harboured four mutant alleles. With these mutations, the total number of mutations in this gene reaches 129. The absence of mutations in 10/22 (45.45%) families suggests that the mutations could be in deep intronic or promoter regions of this gene or these families could have mutations in a yet to be identified gene. The present study increases the mutation landscape of PLA2G6. The present finding will be useful for genetic diagnosis, carrier detection and genetic counselling to families included in this study and other families with similar disease condition.