Genome-Wide Polygenic Score Predicts Large Number of High Risk Individuals in Monogenic Undiagnosed Young Onset Parkinson's Disease Patients from India.

Parkinson's disease (PD) is a genetically heterogeneous neurodegenerative disease with poorly defined environmental influences. Genomic studies of PD patients have identified disease-relevant monogenic genes, rare variants of significance, and polygenic risk-associated variants. In this study, whole genome sequencing data from 90 young onset Parkinson's disease (YOPD) individuals are analyzed for both monogenic and polygenic risk. The genetic variant analysis identifies pathogenic/likely pathogenic variants in eight of the 90 individuals (8.8%). It includes large homozygous coding exon deletions in PRKN and SNV/InDels in VPS13C, PLA2G6, PINK1, SYNJ1, and GCH1. Eleven rare heterozygous GBA coding variants are also identified in 13 (14.4%) individuals. In 34 (56.6%) individuals, one or more variants of uncertain significance (VUS) in PD/PD-relevant genes are observed. Though YOPD patients with a prioritized pathogenic variant show a low polygenic risk score (PRS), patients with prioritized VUS or no significant rare variants show an increased PRS odds ratio for PD. This study suggests that both significant rare variants and polygenic risk from common variants together may contribute to the genesis of PD. Further validation using a larger cohort of patients will confirm the interplay between monogenic and polygenic variants and their use in routine genetic PD diagnosis and risk assessment.

Compound heterozygous variants in Wiskott-Aldrich syndrome like (WASL) gene segregating in a family with early onset Parkinson's disease.

BACKGROUND: Knowledge of genetic determinants in Parkinson's disease is still limited. Familial forms of the disease continue to provide a rich resource to capture the genetic spectrum in disease pathogenesis, and this approach is exploited in this study. METHODS: Informative members from a three-generation family of Indian ethnicity manifesting a likely autosomal recessive mode of inheritance of Parkinson's disease were used for whole exome sequencing. Variant data analysis and in vitro functional characterisation of variant(s) segregating with the phenotype were carried out in HEK-293 and SH-SY5Y cells using gene constructs of interest. RESULTS: Two compound heterozygous variants, a rare missense (c.1139C > T:p.P380L) and a novel splice variant (c.1456 + 2 delTAGA, intron10) in Wiskott-Aldrich syndrome like gene (WASL, 7q31), both predicted to be deleterious were shared among the proband and two affected siblings. WASL, a gene not previously linked to a human Mendelian disorder is known to regulate actin polymerisation via Arp2/3 complex. Based on exon trapping assay using pSPL3 vector in HEK-293 cells, the splice variant showed skipping of exon10. Characterisation of the missense variant in SH-SY5Y cells demonstrated: i) significant alterations in neurite length and number; ii) decreased reactive oxygen species tolerance in mutation carrying cells on Tetrabutylphosphonium hydroxide induction and iii) increase in alpha-synuclein protein. Screening for WASL variants in two independent PD cohorts identified four individuals with heterozygous but none with biallelic variants. CONCLUSION: WASL, with demonstrated functional relevance in neurons may be yet another strong candidate gene for autosomal recessive PD encouraging assessment of its contribution across populations.

Novel and reported variants in Parkinson's disease genes confer high disease burden among Indians.

BACKGROUND: Genetic heterogeneity in Parkinson's disease (PD) has been unambiguously reported across different populations. Assuming a higher genetic load, we tested variant burden in PD genes to an early onset PD cohort from India. METHODS: Whole exome sequencing was performed in 250 PD patients recruited following MDS-UPDRS criteria. The number of rare variants in the 20 known PD genes per exome were used to calculate average rare variant burden with the 616 non-PD exomes available in-house as a comparison group. SKAT-O test was used for gene level analysis. RESULTS: 80 patients harboured rare variants in 20 PD genes, of which six had known pathogenic variants accounting for 2.4% of the cohort. Of 80 patients, 12 had homozygous and nine had likely compound heterozygous variants in recessive PD genes and 59 had heterozygous variants in only dominant PD genes. Of the 16 novel variants of as yet unknown significance identified, four homozygous across ATP13A2, PRKN, SYNJ1 and PARK7; and 12 heterozygous among LRRK2, VPS35, EIF4G1 and CHCHD2 were observed. SKAT-O test suggested a higher burden in GBA (punadjusted = 0.002). Aggregate rare variant analysis including 75 more individuals with only heterozygous variants in recessive PD genes (excluding GBA), with an average of 0.85 protein-altering rare variants per PD patient exome versus 0.51 in the non-PD group, revealed a significant enrichment (p < 0.0001). CONCLUSION: This first study in an early onset PD cohort among Indians identified 16 novel variants in known genes and also provides evidence for a high genetic burden in this ethnically distinct population.

Early Onset Parkinson's disease due to DJ1 mutations: An Indian study.

INTRODUCTION: Early Onset Parkinson's Disease (EOPD) is genetically heterogeneous. PARK2 mutations are the commonest cause of autosomal recessive EOPD followed by PINK1.DJ1 mutations is rare and there is scarce literature on its phenotype and long term outcome. OBJECTIVES: We undertook a retrospective study to determine the prevalence of DJ1 mutation(s) in an Indian population and describe the clinical features and long term outcome of EOPD patients with these mutations. METHODS: One hundred EOPD patients and 114 controls were evaluated. All the seven coding exons of DJ1 gene were screened for novel and reported mutations by PCR- Sanger sequencing. RESULTS: A novel homozygous missense mutation (c.313 A > T, p. Ile105Phe) in exon 5 was seen in one patient and four unrelated patients had a homozygous missense single nucleotide variant rs71653619 (c.293 G > A, p.Arg98Gln). The clinical phenotype comprised of asymmetrical onset, slowly progressive Parkinsonism with levodopa induced motor restlessness in a patient with the novel mutation (c.313 A > T, p. Ile105Phe) while subjects with c.293 G > A, p.Arg98Gln had early onset levodopa responsive symmetrical Parkinsonism. CONCLUSION: DJ1 mutations account for ∼5% of EOPD patients from the Indian population. This study further adds to the clinical spectrum of EOPD with DJ1 mutations.

Evidence of mutations in RIC3 acetylcholine receptor chaperone as a novel cause of autosomal-dominant Parkinson's disease with non-motor phenotypes.

BACKGROUND: The known genetic determinants of Parkinson's disease (PD) do not explain all cases investigated to date. Contemporary sequencing technologies hold promise for enhanced causal variant discovery. We attempted to identify the putative causal variant in an Indian PD family by whole exome sequencing (WES). METHODS: WES data generated for two affected cousins from a 14-member PD family with some non-motor phenotypes were analysed. Variants prioritised were checked for segregation with disease by targeted sequencing. An independent PD cohort (n=280) was screened for additional mutations in the prioritised gene. Variants were functionally validated in PC12 cells differentiated into neurons. RESULTS: A heterozygous mutation c.169C>A, p.P57T in RIC3 acetylcholine receptor chaperone (11p15) segregated with disease in the family confirming an autosomal-dominant mode of inheritance. Another heterozygous mutation c.502G>C, p.V168L was detected in an unrelated PD case. Both mutations were absent in 144 healthy control and in 74 non-PD WES data available in-house and in 186 age and sex-matched controls screened by PCR sequencing. RIC3 is a known chaperone of neuronal nicotinic acetylcholine receptor subunit α-7 (CHRNA7). Dominant negative effect of RIC3 mutants in transfected PC12 cells was reflected by the reduced levels of endogenous CHRNA7 in the membrane fractions in western blots and lower colocalisation profiles in confocal micrographs. CONCLUSION: The novel demonstration of a chaperone-mediated receptor density alteration due to RIC3 mutants provides strong evidence for the role of cholinergic pathway for the first time in PD aetiology. This may also be insightful for some non-motor symptoms and personalised treatment.

Discovery of a frameshift mutation in podocalyxin-like (PODXL) gene, coding for a neural adhesion molecule, as causal for autosomal-recessive juvenile Parkinsonism.

BACKGROUND: Mutations in known genes for inherited forms of Parkinson's disease (PD) account for <30% of familial PD (FPD) implying that more causal gene(s) remain to be identified. We attempted to discover the putative causal variant in an Indian family with autosomal-recessive juvenile Parkinsonism (ARJP), tested negative for mutations in PARK2, PINK1 and DJ1. METHODS: Whole exomes of two affected siblings were sequenced. Variants prioritised were screened for segregation with disease in the family by targeted sequencing. Gene thus identified was screened for index/additional exonic mutations, if any, in an independent PD cohort by PCR sequencing. Variants observed were functionally validated in differentiated PC12 cells. RESULTS: A novel homozygous frameshift mutation, c.89_90insGTCGCCCC in exon 1 of podocalyxin-like gene (PODXL, 7q32-33), resulting in loss of protein, segregated with disease in the family. Mutant allele was absent in 186 healthy controls screened by PCR sequencing and in control exomes available in the laboratory and public databases. Screening of additional 212 sporadic and 68 FPD cases identified three novel heterozygous missense variants namely c.1285C>A, c.1118G>A and c.881G>A in three unrelated cases. Significant differences in neurite branching and length (p<0.0001) were observed in PC12 cells with wild-type and mutant constructs. CONCLUSIONS: Based on the genetic and functional evidence in this study and literature support on the role of PODXL in neural development, a novel frameshift mutation in PODXL seems to be the likely cause of ARJP in this family. This is the first report suggesting the possible role of a neurodevelopmental pathway in PD aetiology.

VPS35 and EIF4G1 mutations are rare in Parkinson's disease among Indians.

Mutations in 2 genes, vacuolar protein sorting homolog 35 (VPS35) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), have been recently reported as causal in autosomal dominant Parkinson's disease (PD) among Caucasians. Their contribution to PD in other ethnic groups remains limited with 1% of VPS35 mutations observed in Caucasian and Japanese populations, but none in Chinese, and 11.57% of EIF4G1 mutations in Caucasian families and 0.09% and 0.17% in Caucasian and Chinese sporadic cases, respectively. We investigated the contribution, if any, of these 2 genes to familial and sporadic PD among the ethnically distinct Indian population. Complete exonic regions of these 2 genes were resequenced in 15 well-characterized PD families; the reported p.Asp620Asn in VPS35 and p.Arg1205His in EIF4G1 mutations were screened in an additional 54 familial and 251 sporadic PD cases, and no mutations were observed. These results, together with our previous reports on the absence of mutations in SNCA and LRRK2, warrant a continuing search for novel causative genes for PD among Indians.

Prevalence and profile of Restless Legs Syndrome in Parkinson's disease and other neurodegenerative disorders: a case-control study.

BACKGROUND: Restless Legs Syndrome (RLS) is associated with impaired central dopaminergic neurotransmission. Though a link between RLS and parkinsonism has been proposed, the prevalence of RLS in parkinsonian disorders is poorly documented. OBJECTIVE: To determine the prevalence of RLS in patients with Parkinson's Disease (PD), Progressive Supranuclear Palsy (PSP), Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). METHODS: We evaluated 187 consecutive patients with parkinsonian disorders (PD = 134, PSP = 27, MSA = 21, DLB = 5) and 172 healthy controls. RLS was diagnosed using the International RLS Study Group (IRLSSG) criteria and the severity of RLS was assessed in patients with definite RLS. Quality of sleep was evaluated with established scales. RESULTS: The prevalence of RLS was higher in patients compared to controls (9.6% vs. 2.9%; p = 0.009) and was highest in PD (11.9%). RLS was present in only one patient each with MSA and PSP and none with DLB. The mean IRLSSG severity score of patients was 16.2 ± 6.5. The global Pittsburgh Sleep Quality Index score and Epworth Sleepiness Scale score were significantly higher in patients compared to controls (p < 0.001). PD patients with RLS had lower Parkinson's Disease Sleep Scale (PDSS) score compared to patients without RLS (p = 0.023). There was no significant difference in gender, age, duration and severity of PD between the two groups. CONCLUSIONS: Our study found a higher prevalence of RLS in PD compared to healthy controls or other parkinsonian disorders. Apart from PDSS score, there was no significant difference in the clinical characteristics of PD patients with and without RLS.

Leads from xenobiotic metabolism genes for Parkinson's disease among north Indians.

OBJECTIVE: Pesticide/neurotoxin/free radical-induced oxidative stress leading to dopaminergic neuronal vulnerability is known to promote sporadic Parkinson's disease (PD). This study investigated the contribution of polymorphisms in genes from drug-metabolizing enzymes (DMEs) and the oxidative stress pathway to PD susceptibility and severity among a north Indian cohort. METHODS: Three hundred and thirty-nine PD patients diagnosed using UK PD brain bank criteria and 344 age-, sex-, and ethnicity-matched controls were recruited. Univariate and multivariate analyses were carried out to test allelic, genotypic, and haplotypic associations, and gene-gene interactions were assessed for 18 polymorphisms from 13 genes. Disease severity was calculated on the basis of the Hoehn and Yahr (HY) scale and Unified Parkinson's Disease Rating Scale III scores and was compared among the genotypic categories of markers. RESULTS: An association of GSTO1-rs4925 (P=0.04) and NQO1-rs1800566 (P=0.02) in univariate and multivariate analysis (P=0.01 and P=0.03, respectively) with disease susceptibility was observed. Significant and novel association of PON2-rs7493 (P=0.00009 with UPDRS III, P=0.003 with HY) with disease severity was retained after Bonferroni correction. On categorizing the cohort into young-onset PD (YOPD, n=90 cases, 104 controls) and late-onset PD ( n=249 cases, 240 controls), the association of several single nucleotide polymorphisms (SNPs) in DMEs was observed with YOPD. CONCLUSIONS: The association of NQO1, PON2, and DME genes (this study) and NAT2 (previous study) with PD among Indians may point toward an inherent population-specific genetic predisposition. This, probably compounded by an increase in environmental toxins and the indiscriminate use of pesticides in our country in the last few decades, may suggest likely gene-environment interactions, which may explain the increasing incidence of YOPD among Indians.

Role of dysautonomic symptoms in distinguishing Parkinson's disease (PD) from multiple system atrophy (MSA-P) within a year of developing motor symptoms.

OBJECTIVE: Can dysautonomic symptoms occurring within a year of developing motor symptoms distinguish Multiple system atrophy-Parkinsonian (MSA-P) from Parkinson's disease (PD)? PATIENTS AND METHODS: Seventy-two Parkinsonian patients diagnosed as probable PD or MSA-P. RESULTS: PD (n = 58, 80.6%) and MSA (n = 14, 19.4%) patients were of similar age and had motor symptoms for similar duration. PD first presents with motor symptoms (68.3%) while MSA-P presents with dysautonomia (85.7%). Urinary incontinence was reported by MSA-P (64%) at their first visit and was absent in most PD (98%) patients. CONCLUSIONS: Urinary incontinence and orthostatic symptoms occurring in a parkinsonian patient within one-year history of motor symptoms suggests a diagnosis of MSA-P with high accuracy and their absence suggests PD.

Evaluation of markers of oxidative stress, antioxidant function and astrocytic proliferation in the striatum and frontal cortex of Parkinson's disease brains.

Dopaminergic neurons die in Parkinson's disease (PD) due to oxidative stress and mitochondrial dysfunction in the substantia nigra (SN). We evaluated if oxidative stress occurs in other brain regions like the caudate nucleus (CD), putamen (Put) and frontal cortex (FC) in human postmortem PD brains (n = 6). While protein oxidation was elevated only in CD (P < 0.05), lipid peroxidation was increased only in FC (P < 0.05) and protein nitration was unchanged in PD compared to controls. Interestingly, mitochondrial complex I (CI) activity was unaffected in PD compared to controls. There was a 3-5 fold increase in the total glutathione (GSH) levels in the three regions (P < 0.01 in FC and CD; P < 0.05 in Put) but activities of antioxidant enzymes catalase, superoxide dismutase, glutathione reductase and glutathione-s-tranferase were not increased. Total GSH levels were elevated in these areas because of decreased activity of gamma glutamyl transpeptidase (γ-GT) (P < 0.05) activity suggesting a decreased breakdown of GSH. There was an increase in expression of glial fibrillary acidic protein (GFAP) (P < 0.001 in FC; P < 0.05 in CD) and glutathione peroxidase (P < 0.05 in CD and Put) activity due to proliferation of astrocytes. We suggest that increased GSH and astrocytic proliferation protects non-SN brain regions from oxidative and mitochondrial damage in PD.

Urogenital symptoms in Parkinson's disease and multiple system atrophy-Parkinsonism: at onset and later.

METHODS: One hundred and eighty-one parkinsonian patients were evaluated to determine if urogenital symptoms at presentation to the Neurology clinic can differentiate them as PD or MSA-P. An autonomic questionnaire was used to document urinary and genital symptoms. RESULTS: Mean age at presentation and disease duration in PD and MSA-P were similar. Urinary symptoms occurred twice as frequently in MSA-P than in PD. Storage symptoms (frequency, urgency, urge incontinence, nocturia) were common in both Parkinsonian disorders. Male MSA-P reported genital symptoms (erectile and ejaculatory failure) three times more frequently than in PD. CONCLUSIONS: Urogenital symptoms occurred in MSA-P when they had mild motor few symptoms unlike in PD where they occur when motor symptoms were severe. Urogenital dysfunction occurred early and was present in all MSA-P patients within two years. Presence of urogenital symptoms in early stages of Parkinsonism strongly favors MSA-P rather than PD. Absence of urogenital symptoms in advanced Parkinsonism makes MSA-P unlikely.

Role of polymorphisms in dopamine synthesis and metabolism genes and association of DBH haplotypes with Parkinson's disease among North Indians.

OBJECTIVES: Genetic and non-genetic components are believed to govern the etiology of common complex traits such as Parkinson's disease (PD). In view of the biochemical evidence of depleted dopamine levels in the affected brains and also the most common and effective therapeutic modality of administration of levodopa in PD, genes from the dopaminergic pathway emerge as major determinants. We have earlier shown the role of DRD4-120 bp duplication marker in PD susceptibility. In this study, contribution of six genes involved in dopamine synthesis and metabolism to PD susceptibility and disease severity was assessed in a North Indian PD cohort. METHODS: 339 patients diagnosed using UKPD brain bank criteria and 344 matched controls were recruited and disease severity was assessed using the Hoehn and Yahr scale and Unified Parkinson Disease Rating Scale III scores. Allelic, genotypic and haplotypic associations with PD were computed; severity was compared among the genotypic categories of markers; gene-gene interactions were assessed using multiple logistic regression. RESULTS: A highly significant association of dopamine beta-hydroxylase (DBH) haplotypes (rs1611115T>C - rs1108580A>G - rs5320A>G - rs129882C>T) with PD was observed; haplotypes C-A-G-C [P=0.000005, Odds ratio (95% confidence interval): OR (95% CI)=1.76 (1.38-2.25)] and C-A-G-T [P=0.000001, OR (95% CI)=0.49 (0.37-0.65)] retaining significance after Bonferroni correction. rs129882, a 3'UTR SNP in DBH showed significant association with disease severity [Hoehn and Yahr (P=0.005) and Unified Parkinson Disease Rating Scale (P=0.006)]. CONCLUSION: Observed association of DBH SNP/SNP haplotypes with PD susceptibility and its role in modulating disease severity reiterates the importance of dopamine pathway in sporadic PD etiology in general and potential therapeutic implications of DBH in particular.

Biotin-responsive basal ganglia disease: a treatable and reversible neurological disorder of childhood.

Biotin-responsive basal ganglia disease is a rare childhood neurological disorder of uncertain etiology that is treatable if suspected and diagnosed. Only few cases have been reported earlier in literature. We report a case of biotin-responsive basal ganglia disease suspected clinically, corroborated by neuroimaging and a dramatic response to biotin therapy.

Are current recommendations to diagnose orthostatic hypotension in Parkinson's disease satisfactory?

We interviewed 50 Parkinson's disease (PD) patients using a questionnaire to verify the reliability of orthostatic symptoms in warning the presence of orthostatic hypotension (OH). OH is defined as 20 mm Hg systolic or 10 mm Hg diastolic BP fall within 3 min of tilting or standing but if this fall occurs after 3 min we called it 'late OH' (L-OH). We compared if OH in Parkinson's disease (PD) was more frequent after head-up tilt or on standing and if the period of postural challenge matters in detecting OH. Twenty-one (42%) patients had OH that occurred twice more often after tilting (n = 20) than on standing (n = 10). OH occurred within 3 min of tilting in 9 patients (18%) and appeared beyond the currently recommended 3 min in 11 patients (55%) (L-OH). Ten of the 20 patients developing OH on tilting were symptomatic. The 10 patients who had OH on standing were asymptomatic. Reporting of symptoms was independent of age or severity of BP fall. Most (90%) patients reporting orthostatic symptoms on standing had OH on tilting for 3 min. Orthostatic symptoms in PD have a high specificity but low sensitivity in predicting OH. In Parkinson's disease OH occurs often after tilting than on standing and is delayed (after 3 min). As OH in PD is often asymptomatic and delayed it could contribute to falls and increase morbidity. We suggest routine evaluation of OH in PD by tilting them longer than the recommended 3 minutes to detect delayed OH.

Primary cardiac cytotoxic T-cell lymphoma presenting with neurological deficits: a case report.

BACKGROUND: Primary cardiac lymphoma is extremely rare in immunocompetent patients. Clinical manifestations vary, and, most often, diagnosis is not made until autopsy. The majority of reported primary cardiac lymphoma cases have been of B-cell origin, while T-cell cardiac lymphomas have been extremely rare. Occasionally, lymphomas and other systemic malignancies clinically present as paraneoplastic neurological syndromes. METHODS: We report a unique case of primary cardiac peripheral T-cell lymphoma of cytotoxic phenotype, clinically presenting with neurological features of external ophthalmoplegia and lower cranial nerve paresis mimicking mitochondrial cytopathy, that was recognized at autopsy. Brain and thoracoabdominal viscera retrieved at autopsy were fixed in 10% buffered formalin and processed for paraffin embedding. In addition to routine histology, immunohistochemistry for immunophenotypic characterization of lymphoma cells was performed. Fresh skeletal muscle was processed for cryosectioning and histochemical staining. RESULTS: On gross examination, the heart showed multiple circumscribed, whitish nodules on both sides. Histological examination of these nodules revealed lymphomatous deposits-cells expressing CD45, CD2, CD3, CD5, CD7, CD8, perforin, and granzyme B. Histological sections from the brain showed foci of demyelination and patchy perivascular lymphoid cell aggregates in leptomeninges and within the parenchyma. These lymphoid cells expressed CD2, CD3, and CD5, with the T cells being predominantly CD4 (CD4:CD8>2), which was unlike the CD8-predominant lymphomatous infiltrate in the heart. Hence, these lymphoid cells in the brain, rather than disseminated lymphoma cells, were considered to be related to the demyelinating process. There was no evidence of lymphomatous deposits in the rest of the viscera examined. CONCLUSION: A diagnosis of primary cardiac peripheral T-cell lymphoma of cytotoxic phenotype clinically manifesting as paraneoplastic demyelinating lesions in the brain was described.