Senthil Selvaraj

Adenylate cyclase 3 (ADCY3) is a transmembrane protein predominantly expressed in the primary cilia of neurons. It plays a vital role in converting ATP to cAMP, a secondary messenger that regulates various downstream signaling pathways such as carbohydrates and lipids metabolism. Homozygous loss-of-function variants in the ADCY3 gene lead to severe early-onset obesity and insulin resistance whereas gain-of-function variants protect against obesity. To describe a novel pathogenic ADCY3 variant implicated in early-onset obesity and functionally characterize this variant via in vitro and in silico validation, we identified a novel homozygous nonsense variant c.2520C>G, p.Thr840X in the ADCY3 gene using gene panel sequencing in a four-year-old girl. She was born to first-cousin consanguineous parents. The patient presented with severe obesity, and exhibited hepatomegaly and insulin resistance, with other biochemical and hormonal tests being normal. In vitro and in silico functional analyses showed downregulation and impaired activation of the ADCY3 protein. Our findings contribute to existing research that supports the role of ADCY3 in the genetic pathogenesis of early-onset obesity. In vitro and in silico functional characterization of the novel p.Thr840X variant showed impaired enzymatic activity leading to receptor loss of function, consistent with the patient’s phenotype. Genetic testing is essential in severe early-onset obesity and early diagnosis could benefit patients with personalized treatment strategies.

Clinical implementation of pharmacogenomics will help in personalizing drug prescriptions and alleviate the personal and financial burden due to inefficacy and adverse reactions to drugs. However, such implementation is lagging in many parts of the world, including the Middle East, mainly due to the lack of data on the distribution of actionable pharmacogenomic variation in these ethnicities. We analyzed 6,045 whole genomes from the Qatari population for the distribution of allele frequencies of 2,629 variants in 1,026 genes known to affect 559 drugs or classes of drugs. We also performed a focused analysis of genotypes or diplotypes of 15 genes affecting 46 drugs, which have guidelines for clinical implementation and predicted their phenotypic impact. The allele frequencies of 1,320 variants in 703 genes affecting 299 drugs or class of drugs were significantly different between the Qatari population and other world populations. On average, Qataris carry 3.6 actionable genotypes/diplotypes, affecting 13 drugs with guidelines for clinical implementation, and 99.5% of the individuals had at least one clinically actionable genotype/diplotype. Increased risk of simvastatin-induced myopathy could be predicted in ~32% of Qataris from the diplotypes of SLCO1B1, which is higher compared to many other populations, while fewer Qataris may need tacrolimus dosage adjustments for achieving immunosuppression based on the CYP3A5 diplotypes compared to other world populations. Distinct distribution of actionable pharmacogenomic variation was also observed among the Qatari subpopulations. Our comprehensive study of the distribution of actionable genetic variation affecting drugs in a Middle Eastern population has potential implications for preemptive pharmacogenomic implementation in the region and beyond.

Despite recent biomedical breakthroughs and large genomic studies growing momentum, the Middle Eastern population, home to over 400 million people, is under-represented in the human genome variation databases. Here we describe insights from phase 1 of the Qatar Genome Program with whole genome sequenced 6,047 individuals from Qatar. We identified more than 88 million variants of which 24 million are novel and 23 million are singletons. Consistent with the high consanguinity and founder effects in the region, we found that several rare deleterious variants were more common in the Qatari population while others seem to provide protection against diseases and have shaped the genetic architecture of adaptive phenotypes. These results highlight the value of our data as a resource to advance genetic studies in the Arab and neighbouring Middle Eastern populations and will significantly boost the current efforts to improve our understanding of global patterns of human variations, human history and genetic contributions to health and diseases in diverse populations.

Calcium (Ca²⁺) functions as a second messenger that is critical in regulating fundamental physiological functions such as cell growth/development, cell survival, neuronal development and/or the maintenance of cellular functions. The coordination among various proteins/pumps/Ca²⁺ channels and Ca²⁺ storage in various organelles is critical in maintaining cytosolic Ca²⁺ levels that provide the spatial resolution needed for cellular homeostasis. An important regulatory aspect of Ca²⁺ homeostasis is a store operated Ca²⁺ entry (SOCE) mechanism that is activated by the depletion of Ca²⁺ from internal ER stores and has gained much attention for influencing functions in both excitable and non-excitable cells. Ca²⁺ has been shown to regulate opposing functions such as autophagy, that promote cell survival; on the other hand, Ca²⁺ also regulates programmed cell death processes such as apoptosis. The functional significance of the TRP/Orai channels has been elaborately studied; however, information on how they can modulate opposing functions and modulate function in excitable and non-excitable cells is limited. Importantly, perturbations in SOCE have been implicated in a spectrum of pathological neurodegenerative conditions. The critical role of autophagy machinery in the pathogenesis of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, would presumably unveil avenues for plausible therapeutic interventions for these diseases. We thus review the role of SOCE-regulated Ca²⁺ signaling in modulating these diverse functions in stem cell, immune regulation and neuromodulation.

Background: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are serious inherited heart diseases with various causative mutations identified. The full spectrum of causative mutations remains to be discovered, especially in understudied populations.Methods: Here, we established the DOHA Registry and Biobank for cardiomyopathies in Qatar, followed by sequencing of 174 genes on 51 HCM and 53 DCM patients, and 31 relatives.Results: In HCM, the analysis of 25 HCM‐associated genes showed that 20% of HCM cases had putative pathogenic variants for cardiomyopathy, mainly in sarcomere genes. Additional 49% of HCM cases had variants of uncertain significance, while 31% of HCM cases had likely benign variant(s) or had no variants identified within the analyzed HCM genes. In DCM, 56 putative DCM genes were analyzed. Eight percent of DCM cases had putative pathogenic variants for DCM, in the TTN gene while 70% of cases had variants of uncertain significance, in the analyzed DCM genes, that will need further pathogenicity assessment. Moreover, 22% of DCM cases remain unexplained, by having likely benign variant(s) or having no variants detected in any of the analyzed DCM genes.Conclusion: We identified or replicated at least four recurrent variants among cardiomyopathy patients, which could be founder disease mutations in the Arabic population, including a frameshift variant (c.1371_1381dupTATCCAGTTAT) of unknown significance in the FKTN gene which seems to cause DCM in homozygosity, and HCM in heterozygosity. In vivo and/or in vitro functional validation need to be pursued in order to assess the pathogenicity of the identified variants.

In the present study, the modulatory effect of ursolic acid (UA) on cardiac fibrosis and mitochondrial and lysosomal enzymes activity in isoproterenol-induced myocardial infarction (MI) in rats were examined. Isoproterenol hydrochloride (ISO; 85 mg/kg body weight) was administered subcutaneously for first two consecutive days. ISO-induced MI in rats significantly decreased the activities of mitochondrial tricarboxylic acid cycle enzymes and respiratory chain enzymes while increased the activities of lysosomal glycohydrolases and cathepsins. The expression of matrix metalloproteinase 2 (MMP-2), MMP-9, collagen type I, α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) were upregulated in ISO-induced MI in rats. UA administration to rats showed increased activities of mitochondrial tricarboxylic acid cycle enzymes and respiratory chain enzymes and decreased activities of lysosomal glycohydrolases and cathepsins in ISO-induced rats. Furthermore, expression of MMP-2, MMP-9, collagen type I, α-SMA, and TGF-β downregulated in UA-administered rats. Thus, our results demonstrate that UA has an anti-fibrotic effect and attenuates the mitochondrial and lysosomal dysfunction in ISO-induced MI in rats.

Parkinson’s disease is a neurodegenerative disorder involving the progressive loss of dopaminergic neurons (DNs), with currently available therapeutics, such as L-Dopa, only able to relieve some symptoms. Stem cell replacement is an attractive therapeutic option for PD patients, and DNs derived by differentiating patient specific stem cells under defined in-vitro conditions may present a viable opportunity to replace dying neurons. We adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DN using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF. While MSC-derived DNs have been characterized for neuronal markers and electrophysiological properties, we investigated store-operated calcium entry (SOCE) mechanisms of these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP⁺). Overall, we show that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP⁺ exposure dysregulates calcium signaling, making them vulnerable to neurodegeneration. Since in-vitro differentiation of MSCs into DNs is an important vehicle for PD disease modeling and regenerative medicine, the results of this study may help with understanding of the pathological mechanisms underlying PD.

Parkinson’s disease (PD) is a common neurodegenerative disorder, which involves degeneration of dopaminergic neurons that are present in the substantia nigra pars compacta (SNpc) region. Many factors have been identified that could lead to Parkinson’s disease; however, almost all of them are directly or indirectly dependent on Ca²⁺ signaling. Importantly, though disturbances in Ca²⁺ homeostasis have been implicated in Parkinson’s disease and other neuronal diseases, the identity of the calcium channel remains elusive. Members of the transient receptor potential canonical (TRPC) channel family have been identified as a new class of Ca²⁺ channels, and it could be anticipated that these channels could play important roles in neurodegenerative diseases, especially in PD. Thus, in this chapter we have entirely focused on TRPC channels and elucidated its role in PD.

Loss of dopaminergic (DA) neurons leads to Parkinson's disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca²⁺channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1^−/−, mice. Similarly, TRPC1^−/−substantia nigra neurons showed increased L-type Ca²⁺currents, decreased stimulation-dependent STIM1-Ca_v1.3 interaction, and decreased DA neurons. L-type Ca²⁺currents and the open channel probability of Ca_v1.3 channels were also reduced upon TRPC1 activation, whereas increased Ca_v1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Ca_v1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, which was prevented by inhibiting L-type Ca²⁺channels. Neurotoxins that mimic Parkinson's disease increased Ca_v1.3 function, decreased TRPC1 expression, inhibited Tg-mediated STIM1-Ca_v1.3 interaction, and induced caspase activation. Importantly, restoration of TRPC1 expression not only inhibited Ca_v1.3 function but increased cell survival. Together, we provide evidence that TRPC1 suppresses Ca_v1.3 activity by providing an STIM1-based scaffold, which is essential for DA neuron survival.SIGNIFICANCE STATEMENTCa²⁺entry serves critical cellular functions in virtually every cell type, and appropriate regulation of Ca²⁺in neurons is essential for proper function. In Parkinson's disease, DA neurons are specifically degenerated, but the mechanism is not known. Unlike other neurons, DA neurons depend on Ca_v1.3 channels for their rhythmic activity. Our studies show that, in normal conditions, the pacemaking activity in DA neurons is inhibited by the TRPC1-STIM1 complex. Neurotoxins that mimic Parkinson's disease target TRPC1 expression, which leads to an abnormal increase in Ca_v1.3 activity, thereby causing degeneration of DA neurons. These findings link TRPC1 to Ca_v1.3 regulation and provide important indications about how disrupting Ca²⁺balance could have a direct implication in the treatment of Parkinson's patients.

In neurons, Ca²⁺ is essential for a variety of physiological processes that regulate gene transcription to neuronal growth and their survival. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ions (MPP⁺) are potent neurotoxins that selectively destroys the dopaminergic (DA) neurons and mimics Parkinson’s disease (PD) like symptoms, but the mechanism as how MPP⁺/MPTP effects DA neuron survival is not well-understood. In the present study, we found that MPP⁺ treatment increased the level of reactive oxygen species (ROS) that activates and upregulates the expression and function of melastatin-like transient receptor potential (TRPM) subfamily member, melastatin-like transient receptor potential channel 2 (TRPM2). Correspondingly, TRPM2 expression was also increased in substantia nigra of MPTP-induced PD mouse model and PD patients. ROS-mediated activation of TRPM2 resulted in an increased intracellular Ca²⁺, which in turn promoted cell death in SH-SY5Y cells. Intracellular Ca²⁺ overload caused by MPP⁺-induced ROS also affected calpain activity, followed by increased caspase 3 activities and activation of downstream apoptotic pathway. On the other hand, quenching of H₂O₂ by antioxidants, resveratrol (RSV), or N-acetylcysteine (NAC) effectively blocked TRPM2-mediated Ca²⁺ influx, decreased intracellular Ca²⁺ overload, and increased cell survival. Importantly, pharmacological inhibition of TRPM2 or knockdown of TRPM2 using siRNA, but not control siRNA, showed an increased protection by preventing MPP⁺-induced Ca²⁺ increase and inhibited apoptosis. Taken together, we show here a novel role for TRPM2 expression and function in MPP⁺-induced dopaminergic neuronal cell death.