Spinocerebellar ataxia type 12 (SCA12) is a progressive late-onset neurodegenerative disorder caused by expansion of ≥ 43 trinucleotide CAG repeats in the upstream non-coding region of the PPP2R2B gene at locus 5q32 (SCA12; OMIM#604326). Clinically SCA12 patients predominately present hand tremor, gait ataxia, tremulous voice and other neurological and psychiatric features. Neuroimaging reveals degenerative changes in the cerebral cortex and cerebellum, however, the underlying disease mechanism at molecular level is still incompletely understood. Here we report generation of four induced pluripotent stem cells (iPSCs) of SCA12 patients. The established lines were positive for PPP2R2B-CAG expansion mutation and showed expression of undifferentiated hPSC state markers, three germ layer differentiation potential, normal genetic integrity and contamination-free culture.

Gallbladder cancer (GBC) is a rare but aggressive biliary tract malignancy. This study explores the transcriptomic profile of GBC to identify differentially expressed genes (DEGs) and dysregulated pathways involved in its pathogenesis. RNA sequencing was performed on 13 GBC tumors and 6 matched controls. Functional enrichment analysis (FEA) as well as weighted gene co-expression network analysis (WGCNA) were used to identify dysregulated pathways, functionally relevant gene modules and hub genes. Key targets were validated in patient tissues and cell lines. A total of 1319 DEGs were identified (528 upregulated, 791 downregulated). Gene set enrichment analysis revealed activation of E2F targets and G2/M checkpoint, with downregulation of bile acid metabolism and estrogen response pathways. A tumor grade-correlated gene module was identified by WGCNA. FEA of the gene module highlighted pathways related to cell cycle and cell division. Co-expression analysis identified TPX2 as a central hub gene. Inhibitors of aurora kinase, TPX2 dependent enzyme, significantly reduced proliferation, migration, and invasion in GBC cells. Elevated Aurora kinases expression was also observed in GBC. This first transcriptomic analysis of GBC in South-East Asian Indians uncovers key drivers like TPX2 and Aurora kinases in disease progression. The study highlights cell cycle dysregulation and sex-linked signatures, offering insights for biomarker discovery and targeted therapies.

Effective mutagenic treatment techniques for different species are of tremendous interest due to the exciting potential of mutation breeding in ornamental plants. The present article addresses the mutagenesis treatments of numerous ornamental genera, the benefits and drawbacks of different methods, and the potential for enhancing the related protocols. There are several techniques for non-targeted mutagenesis, from chemical treatment with alkylating chemicals to dose-dependent exposure to X-rays, gamma rays, neutron or heavy ion beams. All of these have been shown to be efficient mutagens in a wide range of different species and are reasonably priced. However, due to the high cost and lack of understanding required to efficiently transform and regenerate attractive crops, genetic engineering is still generally impracticable for many ornamental breeding operations. The most widely used non-targeted mutagen currently in use is gamma radiation. Although it appears to have a lower mutagenic efficacy than chemical mutagens, it offers excellent consistency. Although chronic irradiation over a longer period of time induces less harmful mutations than the routinely employed acute irradiation protocols, changes in the radiation dose rate may boost the efficiency. Because of the high particle energy associated with these treatments, heavy ion beam irradiation may also offer extremely consistent mutation induction at greater efficiencies. Additionally, there are chances to enhance chemical mutagenesis. It is still highly beneficial to use mutation breeding, and there are plenty of chances to make the current techniques better.

Spinocerebellar Ataxia type -12 (SCA12) is a neurodegenerative disease caused by tandem CAG repeat expansion in the 5 0 -UTR/non-coding region of PPP2R2B . Molecular pathology of SCA12 has not been studied in the context of CAG repeats, and no appropriate models exist. We found in human SCA12-iPSCderived neuronal lineage that expanded CAG in PPP2R2B transcript forms nuclear RNA foci and were found to sequester variety of proteins. Further, the ectopic expression of transcript containing varying length of CAG repeats exhibits non -canonical repeat -associated non-AUG (RAN) translation in multiple frames in HEK293T cells, which was further validated in patient -derived neural stem cells using specific antibodies. mRNA sequencing of the SCA12 and control neurons have shown a network of crucial transcription factors affecting neural fate, in addition to alteration of various signaling pathways involved in neurodevelopment. Altogether, this study identifies the molecular signatures of SCA12 disorder using patient -derived neuronal cell lines.

Summary Spinocerebellar ataxia type-12 (SCA-12) is a neurodegenerative disease caused by tandem CAG repeat expansion in the 5’-UTR/non-coding region of PPP2R2B . Molecular pathology of SCA12 has not been studied in the context of CAG repeats and no appropriate models exist. We found in human SCA12-iPSC derived neuronal lineage that expanded CAG in PPP2R2B transcript forms nuclear RNA foci and were found to sequester variety of proteins. Further, the ectopic expression of transcript containing varying length of CAG repeats exhibits non-canonical Repeat Associated Non-AUG (RAN) translation in multiple frames in HEK293T cells, which was further validated in patient-derived neural stem cells using specific antibodies. mRNA sequencing of the SCA12 and control neurons have shown a network of crucial transcription factors affecting neural fate, in addition to alteration of various signaling pathways involved in neurodevelopment. Altogether, this study identifies the molecular signatures of spinocerebellar ataxia type-12 disorder using patient-derived neuronal cell lines.

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.

Human Kinesin Family Member 5A (KIF5A) gene mutations have been identified as a putative genetic cause of amyotrophic lateral sclerosis (ALS). Disease modelling using human-induced pluripotent stem cells (HiPSCs) is the next-generation approach to studying numerous human diseases. For the current investigation, we report the generation of patient-specific KIF5A iPSC lines with a mutation at the splice site mutation (c.3020 + 3 A > T) in the intronic region. The resulting line displayed markers for pluripotency, a healthy karyotype, the ability to differentiate into three germ layers in vitro, vector clearance, the KIF5A mutation, STR-based genomic identity, and contamination-free culture.

X-linked hypophosphatemia (XLH), an inheritable form of rickets is caused due to mutation in Phex gene. Several factors are linked to the disease’s aetiology, including non-coding RNA molecules (miRNAs), which are key post-transcriptional regulators of gene expression and play a significant role in osteoblast functions. MicroRNAs sequence analysis showed differentially regulated miRNAs in phex silenced osteoblast cells. In this article, we report miR-539-3p, an unidentified novel miRNA, in the functional regulation of osteoblast. MiR-539-3p overexpression impaired osteoblast differentiation. Target prediction algorithm and experimental confirmation by luciferase 3’ UTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over expression of miR-539-3p in osteoblasts down regulated Wnt/beta catenin signaling components and deteriorated trabecular microarchitecture leading to decreased bone formation in ovariectomized (Ovx) mice. Additionally, biochemical bone resorption markers like CTx and Trap-5b were elevated in serum samples of mimic treated group, while, reverse effect was observed in anti-miR treated animals along with increased bone formation marker P1NP. Moreover, transcriptome analysis with miR-539-3p identified a novel uncharacterized Akap-3 gene in osteoblast cells, knock down of which resulted in downregulation of osteoblast differentiation markers at both transcriptional and translational level. Overall, our study for the first time reported the role of miR-539-3p in osteoblast functions and its downstream Akap-3 signalling in regulation of osteoblastogenesis.

The advent of personalised medicine promises a deeper understanding of mechanisms and therefore therapies. However, the connection between genomic sequences and clinical treatments is often unclear. We studied 50 breast cancer patients belonging to a population-cohort in the state of Qatar. From Sanger sequencing, we identified several new deleterious mutations in the estrogen receptor 1 gene (ESR1). The effect of these mutations on drug treatment in the protein target encoded by ESR1, namely the estrogen receptor, was achieved via rapid and accurate protein-ligand binding affinity interaction studies which were performed for the selected drugs and the natural ligand estrogen. Four nonsynonymous mutations in the ligand-binding domain were subjected to molecular dynamics simulation using absolute and relative binding free energy methods, leading to the ranking of the efficacy of six selected drugs for patients with the mutations. Our study shows that a personalised clinical decision system can be created by integrating an individual patient's genomic data at the molecular level within a computational pipeline which ranks the efficacy of binding of particular drugs to variant proteins.

Fusion transcripts can contribute to diversity of molecular networks in the human cortex. In this study, we explored the occurrence of fusion transcripts in normal human cortex along with single neurons and astrocytes. We identified 1305 non-redundant fusion events from 388 transcriptomes representing 59 human cortices and 329 single cells. Our results indicate while the majority of fusion transcripts in human cortex are intra-chromosomal (85%), events found in single neurons and astrocytes were primarily inter-chromosomal (80%). The number of fusions in single neurons was significantly higher than that in single astrocytes (p < 0.05), indicating fusion as a possible contributor towards transcriptome diversity in neuronal cells. The identified fusions were largely private and 4 specific recurring events were found both in cortex and in single neurons but not in astrocytes. We found a significant increase in the number of fusion transcripts in human brain with increasing age both in single cells and whole cortex (p < 0.0005 and < 0.005, respectively). This is likely one of the many possible contributors for the inherent plasticity of the adult brain. The fusion transcripts in fetal brain were enriched for genes for long-term depression; while those in adult brain involved genes enriched for long-term potentiation pathways. Our findings demonstrate fusion transcripts are naturally occurring phenomenon spanning across the health-disease continuum, and likely contribute to the diverse molecular network of human brain.

BACKGROUND:C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. In humans, biallelic variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux, while heterozygous variants in NPR2 (natriuretic peptide receptor 2) and NPPC (natriuretic peptide precursor C), encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the protein kinase cGMP-dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been clearly demonstrated in several animal models.METHODS:Exome sequencing was performed in two girls with severe short stature due to acromesomelic limb shortening, brachydactyly, mild to moderate platyspondyly and progressively increasing metaphyseal alterations of the long bones. Functional characterisation was undertaken for the identified variants.RESULTS:Two homozygous PRKG2 variants, a nonsense and a frameshift, were identified. The mutant transcripts are exposed to nonsense-mediated decay and the truncated mutant cGKII proteins, partially or completely lacking the kinase domain, alter the downstream mitogen activation protein kinase signalling pathway by failing to phosphorylate c-Raf 1 at Ser43 and subsequently reduce ERK1/2 activation in response to fibroblast growth factor 2. They also downregulate COL10A1 and upregulate COL2A1 expression through SOX9.CONCLUSION:In conclusion, we have clinically and molecularly characterised a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP).

Spinocerebellar ataxia type 12 (SCA12) is a progressive neurological disorder with a unique prevalence in North Indian population. Trinucleotide CAG repeat expansion beyond certain threshold (>43 repeats) in the upstream region of PPP2R2B gene is associated with cerebello-cortical atrophy in disease affected individuals. Patients with SCA12 predominantly manifest unique distinguishable feature of early slow and progressive action tremor in upper extremities followed by other variable symptoms such as mild to moderate gait ataxia, speech disturbances with tremulous voice, head tremor, and autonomic abnormalities. At present, there is no definite treatment available to cure this disease and the underlying disease mechanism at molecular level largely remains undetermined. This review focuses on epidemiology, clinico-genetic advancements, and therapeutics interventions emerged over the time in this field.

Objectives: To genetically investigate a hereditary neurodegenerative disease in a kindred with an intra-familial variable phenotype of complicated hereditary spastic paraplegia with and without cerebellar ataxia and motor neuron disease.Methods: With a detailed clinical evaluation, five members of a family with autosomal dominant inherited neurological illness were subjected for whole exome sequencing. The identified disease causing variation was validated functionally by demonstration of exon skipping on cDNA based amplification of KIF5A.Results: We have identified a kindred with multiple affected individuals who manifested complicated hereditary spastic paraplegia (proband), a maternal uncle manifested Amyotrophic lateral sclerosis/Ataxia phenotype and another symptomatic individuals with cerebellar ataxia phenotype. Genetic investigations by whole exome sequencing revealed a novel heterozygous c.3020+3 A>T variant in KIF5A gene segregating with only affected individuals. The variant resides near 3’splice site junction of exon 27 of KIF5A gene. KIF5A transcript was demonstrated to be have skipped exon 27 in RNA extracted from peripheral whole blood.Conclusions: It has also been documented that C-terminal variants of KIF5A are exclusively associated with the ALS phenotype. We reported a family carrying a C-term mutation with a very complex phenotype affecting spinal cord and cerebellar axis, which is contrary to the reported observation with other C-terminal KIF5A mutant allele. This findings allows an understanding of the role of this important family of gene to have a converging molecular alterations leading to Neurodegenration.

To the Editor: Genetic defects in KIF5A gene has been implicated in wide range of neurodegenerative phenotype, length-dependent axonopathies, that is, hereditary spastic paraplegia (SPG10) and Charcot-MarieTooth disease (CMT2), and motor neuron disease. It has been hypothesized that location of KIF5A variants are determinant of this differential phenotypic manifestations. N-terminal mutations affects its motor domain and thus microtubule bindings chiefly and are linked to HSP and CMT phenotype, while C-terminal region may affect its cargo-binding region, thus affects axonal transport of organelles and other cargo proteins are linked to MND phenotype. We report a family with a complicated and variable neurological phenotype in the form of HSP, cerebellar ataxia and ALS (Figure 1B). In brief, proband of the family, a 46 years old gentleman (IV:2, pedigree chart, Figure 1B), started to have walking imbalance at the age of 42 years and with the disease progression his symptoms evolved with development of dysarthria and slowness while writing. There was a history of multiple affected members of the family in maternal lineage (pedigree chart, Figure 1B). Proband's two maternal uncle died at the age of 70 (III:4) and 57 (III:6) years. Patient III:4, had shown symptoms of walking unsteadiness at the of age 57 years and was diagnosed as a case of progressive cerebellar ataxia. While, another affected III:6, manifested the disease at the age of 32 years with symptoms of tremulousness in hands, was initially diagnosed as a case of motor neuron disease at the age of 40 years at AIIMS. At the age of 46 years he was reevaluated for the development of cerebellar features from the age of 43 years. While a 48 year old sibling of proband an early-symptomatic individual (IV:1) of the family, had developed walking imbalance at the age of 46 years. Detailed examination of all the affected showed Proband having a phenotype of complicated HSP, III:6 (deceased) had MND and Ataxia; III:4 (deceased) had cerebellar ataxia and IV:1 has cerebellar ataxia phenotype. Radiological brain examination of affected members had shown variable degree of cerebellar involvement (mild cerebellar atrophy, proband) and as per records (images unavailable) III:4 and III:6 also have shown variable degree of cerebellar degeneration. For genetic investigation, whole-exome sequencing (Illumina Truseq expanded exome) was performed (after ruling out common SCA panel mutations, ie SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17 and SCA36 mutations) for five individuals of the family (pedigree, Figure 1). Initial-targeted WES analysis of gene panel (n = 3197) of candidate loci of neurodegenrative phenotype encompassing, cerebellar ataxia and hereditary spastic paraplegia (https://doi.org/10.6084/m9.figshare.8320223.v1) identified a total of 7815 variants after applying filters for disease model segregating alleles (autosomal dominant inheritance) and frequency cutoff of <0/1% and after removing synonymous calls, 16 variants were left for the correlation analysis. By doing the clinical correlation analysis, a clinically significant intronic variant in KIF5A, a heterozygous, c.3020+3 A>T change which was predicted to be a potentially splice site change was identified to be segregating with the affected individuals only. This was a novel DNA variant in KIF5A; a variant, c.3020+3 A>G (NM_004984) at the same site has been reported by Nicolas et al and was not reported in 1000 Genome, ExAC and gNOMAD dataset and also it was absent in our in-house cohort of 300 exomes of Indian individuals referred for various genetic diseases. This variant is located near 30 donor splice site of the exon 27 of KIF5A. The predicted effect of c.3020+3 A>T was validated for exon skipping from RNA extracted from peripheral blood sample of the proband as described earlier, Figure 1. The exon skipped transcript of KIF5A was predicted to give aberrant C-terminal amino acid residues. KIF5A, is a Kinseins family of protein that exert it chief function with cytoskeletal protein interaction, axonal transport of cellular organalles, that is, mitochondria, neurofilaments and other neuronally expressed important molecules (AMPA and GABAA receptors). 2,3 The phenotype of multiple affected in this family with a c.3023+3 A>T resembled both variable manifestations of complicated HSP phenotype and or cerebellar ataxia and motor neuron disease which later manifested with cerebellar ataxia phenotype. The age at onset of the disease in our presented family is slightly younger to the reported age at onset of c.3020+3 A>G with pure ALS of 50 years, while other C-terminal-associated KIF5A variants exhibit median age at onset of 46.5 years. Overall, this highlights not only the role of modifiers of the disease in this family but also a central role of KIF5A in controlling the variable degree of neuronal degeneration affecting spinal cord to cerebellar axis. Further follow-up of the family members and detailed functional consequences and mechanism of the KIF5A may shed light upon converging pathways of neurodegeneration.

Cerebellar ataxias are a group of rare progressive neurodegenerative disorders with an average prevalence ranges from 4.8 to 13.8 in 100,000 individuals. The inherited disorders affect multiple members of the families, or a community that is endogamous or consanguineous. Presence of more than 3000 mutations in different genes with overlapping clinical symptoms, genetic anticipation and pleiotropy, as well as incomplete penetrance and variable expressivity due to modifiers pose challenges in genotype–phenotype correlation. Development of a diagnostic algorithm could reduce the time as well as cost in clinicogenetic diagnostics and also help in reducing the economic and social burden of the disease. In a unique research collaboration spanning over 20 years, we have been able to develop a paradigm for studying cerebellar ataxias in the Indian population which would also be relevant in other rare diseases. This has involved clinical and genetic analysis of thousands of families from diverse Indian populations. The extensive resource on ataxia has led to the development of a clinicogenetic algorithm for cost-effective screening of ataxia and a unique ataxia clinic in the tertiary referral centre in All India Institute of Medical Sciences. Utilizing a population polymorphism scanning approach, we have been able to dissect the mechanisms of repeat instability and expansion in many ataxias, and also identify founders, and trace the mutational histories in the Indian population. This provides information for genetic testing of at—risk as well as protected individuals and populations. To dissect uncharacterized cases which comprises more than 50% of the cases, we have explored the potential of next-generation sequencing technologies coupled with the extensive resource of baseline data generated in-house and other public domains. We have also developed a repository of patient-derived peripheral blood mononuclear cells, lymphoblastoid cell lines and neuronal lineages (derived from iPSCs) for ascribing functionality to novel genes/mutations. Through integrating these technologies, novel genes have been identified that has broadened the diagnostic panel, increased the diagnostic yield to over 75%, helped in ascribing pathogenicity to novel mutations and enabled understanding of disease mechanisms. It has also provided a platform for testing novel molecules for amelioration of pathophysiological phenotypes. This review through a perspective on CAs suggests a generic paradigm from diagnostics to therapeutic interventions for rare disorders in the context of heterogeneous Indian populations.

Spinocerebellar ataxia type-12 (SCA12) is a neurological disorder caused due to triplet (CAG) repeat expansion in 5' UTR of PPP2R2B. It is one of the most prominent SCA-subtype in Indian population and till date no patient specific models have been described. Human-induced-pluripotent-stem cell (HiPSC) based disease modelling has become the next generation tool for studying various human pathologies. In the present study we established three SCA12 patient specific iPSC lines. All the generated lines have shown pluripotency markers, normal karyotype, in-vitro three germ layers differentiation potential, vector clearance, SCA12 mutation, parental genomic identity and contamination free culture.

Spinocerebellar ataxia type-12 (SCA12) is a neurological disorder that exhibits a unique progressive tremor/ataxia syndrome induced by triplet (CAG) repeat expansion in 5’ UTR of PPP2R2B . SCA12 is one of the most prominent SCA-subtype in India and till date no appropriate disease models have been described. Our aim was to establish human iPSC derived neuronal cell lines of SCA12 and study transcriptomic level alterations induced by CAG expansion. For translational application, peripheral blood transcriptomics of SCA12 patients was also performed. Lymphoblastoid cell lines of three SCA12 patients were reprogrammed to iPSCs and then re-differentiated into pan-neuronal lineage. RNA-sequencing based comparative transcriptomics was performed for disease and control cell lineages. Microarray based transcriptomic profiling of peripheral blood of SCA12 patients was performed in a case/control (n=15/9) design. We have successfully created human neuronal cell lines of SCA12 patient as exhibited by their molecular profiling. Differential expression analysis of RNA-Seq data has shown enrichment for type-I interferon signaling and other relevant cellular processes in SCA12-neurons. At the splice-isoform level, we observed an upregulation of expanded CAG containing non-coding transcript of PPP2R2B . Peripheral blood transcriptomics analysis and targeted validation of RNA-Seq data has allowed us to identify inflammatory signatures as potential markers of molecular pathology in SCA12. Our study has allowed us to establish first iPSC based neuronal cell lines of SCA12. We have identified pro-inflammatory signatures in SCA12-neurons suggestive of a dsRNA mediated activation of interferon signaling and that corroborates with the emerging evidence of neuronal atrophy due to neuro-inflammation in common neurodegenerative diseases. This study involved development of an iPSCs derived neuronal cells of SCA12 and look through signatures of neurodegeneration by whole RNA sequencing. This model sheds light upon key role of RNA mediated induced response in Interferon signaling for neurodegeneration.

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.