New Insights of Phospholipase A2 Associated Neurodegeneration Phenotype Based on the Long-Term Follow-Up of a Large Hungarian Family.

INTRODUCTION: Phospholipase A2-associated Neurodegeneration (PLAN) is a group of neurodegenerative diseases associated with the alterations of PLA2G6. Some phenotype-genotype association are well known but there is no clear explanation why some cases can be classified into distinct subgroups, while others follow a continuous clinical spectrum. METHODS: Long-term neurological, and psychiatric follow-up, neuropathological, radiological, and genetic examinations, were performed in three affected girls and their family. RESULTS: Two 24-years old twins and their 22-years old sister harbored the p.P622S, and p.R600W mutation in PLA2G6. The age of onset and the most prominent presenting symptoms (gaze palsy, ataxia, dystonia, psychomotor regression indicated atypical neuroaxonal dystrophy (ANAD), however, optic atrophy, severe tetraparesis would fit into infantile neuroaxonal dystrophy (INAD). All siblings had hyperintensity in the globi pallidi and substantiae nigrae which is reported in ANAD, whereas it is considered a later neuroradiological marker in INAD. The slow progression, rigidity, bradykinesis, and the prominent psychiatric symptoms indicate PLA2G6-related dystonia-parkinsonism. Abnormal mitochondria, lipid accumulation and axonal spheroids were observed in the muscle and nerve tissue. Brain deposition appeared 6 years following the initial cerebellar atrophy. Mild MRI alterations were detected in the asymptomatic carrier parents. CONCLUSION: The colorful clinical symptoms, the slightly discordant phenotype, and the neuroimaging data in the family supports the view that despite the distinct definition of age-related phenotypes in PLAN, these are not strict disease categories, but rather a continuous phenotypic spectrum. The mild MRI alterations of the parents and the family history suggest that even heterozygous pathogenic variants might be associated with clinical symptoms, although systematic study is needed to prove this.

The Role of the Rare Variants in the Genes Encoding the Alpha-Ketoglutarate Dehydrogenase in Alzheimer's Disease.

There is increasing evidence that several mitochondrial abnormalities are present in the brains of patients with Alzheimer's disease (AD). Decreased alpha-ketoglutarate dehydrogenase complex (αKGDHc) activity was identified in some patients with AD. The αKGDHc is a key enzyme in the Krebs cycle. This enzyme is very sensitive to the harmful effect of reactive oxygen species, which gives them a critical role in the Alzheimer and mitochondrial disease research area. Previously, several genetic risk factors were described in association with AD. Our aim was to analyze the associations of rare damaging variants in the genes encoding αKGDHc subunits and AD. The three genes (OGDH, DLST, DLD) encoding αKGDHc subunits were sequenced from different brain regions of 11 patients with histologically confirmed AD and the blood of further 35 AD patients. As a control group, we screened 134 persons with whole-exome sequencing. In all subunits, a one-one rare variant was identified with unknown significance based on American College of Medical Genetics and Genomics (ACMG) classification. Based on the literature research and our experience, R263H mutation in the DLD gene seems likely to be pathogenic. In the different cerebral areas, the αKGDHc mutational profile was the same, indicating the presence of germline variants. We hypothesize that the heterozygous missense R263H in the DLD gene may have a role in AD as a mild genetic risk factor.

Novel dominant MPAN family with a complex genetic architecture as a basis for phenotypic variability.

OBJECTIVE: Our aim was to study a Hungarian family with autosomal dominantly inherited neurodegeneration with brain iron accumulation (NBIA) with markedly different intrafamilial expressivity. METHODS: Targeted sequencing and multiplex ligation-dependent probe amplification (MLPA) of known NBIA-associated genes were performed in many affected and unaffected members of the family. In addition, a trio whole-genome sequencing was performed to find a potential explanation of phenotypic variability. Neuropathologic analysis was performed in a single affected family member. RESULTS: The clinical phenotype was characterized by 3 different syndromes-1 with rapidly progressive dystonia-parkinsonism with cognitive deterioration, 1 with mild parkinsonism associated with dementia, and 1 with predominantly psychiatric symptoms along with movement disorder. A heterozygous stop-gain variation in the C19Orf12 gene segregated with the phenotype. Targeted sequencing of all known NBIA genes, and MLPA of PLA2G6 and PANK2 genes, as well as whole-genome sequencing in a trio from the family, revealed a unique constellation of oligogenic burden in 3 NBIA-associated genes (C19Orf12 p.Trp112Ter, CP p.Val105PhefsTer5, and PLA2G6 dup(ex14)). Neuropathologic analysis of a single case (39-year-old man) showed a complex pattern of alpha-synucleinopathy and tauopathy, both involving subcortical and cortical areas and the hippocampus. CONCLUSIONS: Our study expands the number of cases reported with autosomal dominant mitochondrial membrane protein-associated neurodegeneration and emphasizes the complexity of the genetic architecture, which might contribute to intrafamilial phenotypic variability.

The Role of Genetic Testing in the Clinical Practice and Research of Early-Onset Parkinsonian Disorders in a Hungarian Cohort: Increasing Challenge in Genetic Counselling, Improving Chances in Stratification for Clinical Trials.

The genetic analysis of early-onset Parkinsonian disorder (EOPD) is part of the clinical diagnostics. Several genes have been implicated in the genetic background of Parkinsonism, which is clinically indistinguishable from idiopathic Parkinson's disease. The identification of patient's genotype could support clinical decision-making process and also track and analyse outcomes in a comprehensive fashion. The aim of our study was to analyse the genetic background of EOPD in a Hungarian cohort and to evaluate the clinical usefulness of different genetic investigations. The age of onset was between 25 and 50 years. To identify genetic alterations, multiplex ligation-dependent probe amplification (n = 142), Sanger sequencing of the most common PD-associated genes (n = 142), and next-generation sequencing (n = 54) of 127 genes which were previously associated to neurodegenerative disorders were carried out. The genetic analysis identified several heterozygous damaging substitutions in PD-associated genes (C19orf12, DNAJC6, DNAJC13, EIF4G1, LRRK2, PRKN, PINK1, PLA2G6, SYNJ1). CNVs in PRKN and SNCA genes were found in five patients. In our cohort, nine previously published genetic risk factors were detected in three genes (GBA, LRRK2, and PINK1). In nine cases, two or three coexisting pathogenic mutations and risk variants were identified. Advances of sequencing technologies make it possible to aid diagnostics of PD by widening the scope of analysis to genes which were previously linked to other neurodegenerative disorders. Our data suggested that rare damaging variants are enriched versus neutral variants, among PD patients in the Hungarian population, which raise the possibility of an oligogenic effect. Heterozygous mutations of multiple recessive genes involved in the same pathway may perturb the molecular process linked to PD pathogenesis. Comprehensive genetic assessment of individual patients can rarely reveal monogenic cause in EOPD, although it may identify the involvement of multiple PD-associated genes in the background of the disease and may facilitate the better understanding of clinically distinct phenocopies. Due to the genetic complexity of the disease, genetic counselling and management is getting more challenging. Clinical geneticist should be prepared for counselling of patients with coexisting disease-causing mutations and susceptibility factors. At the same time, genomic-based stratification has increasing importance in future clinical trials.

Dynamic interaction of genetic risk factors and cocaine abuse in the background of Parkinsonism - a case report.

BACKGROUND: Parkinsonism is a complex multifactorial neurodegenerative disorder, in which genetic and environmental risk factors may both play a role. Among environmental risk factors cocaine was earlier ambiguously linked to Parkinsonism. Former single case reports described Parkinsonism in chronic cocaine users, but an epidemiological study did not confirm an increased risk of Parkinson's disease. Here we report a patient, who developed Parkinsonism in young age after chronic cocaine use, in whom a homozygous LRRK2 risk variant was also detected. CASE PRESENTATION: The patient was investigated because of hand tremor, which started after a 1.5-year period of cocaine abuse. Neurological examination suggested Parkinsonism, and asymmetrical pathology was confirmed by the dopamine transporter imaging study. The genetic investigations revealed a homozygous risk allele in the LRRK2 gene. After a period of cocaine abstinence, the patient's symptoms spontaneously regressed, and the dopamine transporter imaging also returned to near-normal. CONCLUSIONS: This case report suggests that cocaine abuse indeed might be linked to secondary Parkinsonism and serves as an example of a potential gene-environmental interaction between the detected LRRK2 risk variant and cocaine abuse. The reversible nature of the DaTscan pathology is a unique feature of this case, and needs further evaluation, whether this is incidental or can be a feature of cocaine related Parkinsonism.

Comprehensive Analysis of Rare Variants of 101 Autism-Linked Genes in a Hungarian Cohort of Autism Spectrum Disorder Patients.

BACKGROUND: Autism spectrum disorder (ASD) is genetically and phenotypically heterogeneous. Former genetic studies suggested that both common and rare genetic variants play a role in the etiology. In this study, we aimed to analyze rare variants detected by next generation sequencing (NGS) in an autism cohort from Hungary. METHODS: We investigated the yield of NGS panel sequencing of an unselected ASD cohort (N = 174 ) for the detection of ASD associated syndromes. Besides, we analyzed rare variants in a common disease-rare variant framework and performed rare variant burden analysis and gene enrichment analysis in phenotype based clusters. RESULTS: We have diagnosed 13 molecularly proven syndromic autism cases. Strongest indicators of syndromic autism were intellectual disability, epilepsy or other neurological plus symptoms. Rare variant analysis on a cohort level confirmed the association of five genes with autism (AUTS2, NHS, NSD1, SLC9A9, and VPS13). We found no correlation between rare variant burden and number of minor malformation or autism severity. We identified four phenotypic clusters, but no specific gene was enriched in a given cluster. CONCLUSION: Our study indicates that NGS panel gene sequencing can be useful, where the clinical picture suggests a clinically defined syndromic autism. In this group, targeted panel sequencing may provide reasonable diagnostic yield. Unselected NGS panel screening in the clinic remains controversial, because of uncertain utility, and difficulties of the variant interpretation. However, the detected rare variants may still significantly influence autism risk and subphenotypes in a polygenic model, but to detect the effects of these variants larger cohorts are needed.

[Significance of whole exome sequencing in the diagnostics of rare neurological diseases - own experiences through a case presenting with ataxia]. / A teljesexom-szekvenálás jelentosége a ritka neurológiai betegségek diagnosztikájában ­ saját tapasztalatok egy ataxiás eset kapcsán.

Next generation sequencing (NGS) technologies reshape the diagnostics of rare neurological diseases. In the background of certain neurological symptoms, such as ataxia, many acquired and genetic causes may be present. Variations in a given gene can present with variable phenotypes, too. Because of this phenomenon, the conventional one gene sequencing approach often fails to identify the genetic background of a disease. Next generation sequencing panels allow to sequence 50-100 genes simultaneously, and if the disease stratification is not possible based on the clinical symptoms, whole exome sequencing can help in the diagnostic of genetic disorders with atypical presentation. This case study is about the exome sequencing of a patient with cerebellar ataxia. Genetic investigations identified rare variants in the SPG11 gene in association with the clinical phenotype, which gene was originally described in the background of hereditary spastic paraparesis. Our article highlights that in certain cases the variability of the leading presenting symptom makes it hard to select the correct gene panel. In our case the variants in the gene, formerly associated to hereditary spastic paraparesis, resulted in cerebellar ataxia initially, so even an ataxia NGS gene panel would not detect those. Orv Hetil. 2018; 159(28): 1163-1169.

Mitochondrial dysfunction and autism: comprehensive genetic analyses of children with autism and mtDNA deletion.

BACKGROUND: The etiology of autism spectrum disorders (ASD) is very heterogeneous. Mitochondrial dysfunction has been described in ASD; however, primary mitochondrial disease has been genetically proven in a small subset of patients. The main goal of the present study was to investigate correlations between mitochondrial DNA (mtDNA) changes and alterations of genes associated with mtDNA maintenance or ASD. METHODS: Sixty patients with ASD and sixty healthy individuals were screened for common mtDNA mutations. Next generation sequencing was performed on patients with major mtDNA deletions (mtdel-ASD) using two gene panels to investigate nuclear genes that are associated with ASD or are responsible for mtDNA maintenance. Cohorts of healthy controls, ASD patients without mtDNA alterations, and patients with mitochondrial disorders (non-ASD) harbouring mtDNA deletions served as comparison groups. RESULTS: MtDNA deletions were confirmed in 16.6% (10/60) of patients with ASD (mtdel-ASD). In 90% of this mtdel-ASD children we found rare SNVs in ASD-associated genes (one of those was pathogenic). In the intergenomic panel of this cohort one likely pathogenic variant was present. In patients with mitochondrial disease in genes responsible for mtDNA maintenance pathogenic mutations and variants of uncertain significance (VUS) were detected more frequently than those found in patients from the mtdel-ASD or other comparison groups. In healthy controls and in patients without a mtDNA deletion, only VUS were detected in both panel. CONCLUSIONS: MtDNA alterations are more common in patients with ASD than in control individuals. MtDNA deletions are not isolated genetic alterations found in ASD; they coexist either with other ASD-associated genetic risk factors or with alterations in genes responsible for intergenomic communication. These findings indicate that mitochondrial dysfunction is not rare in ASD. The occurring mtDNA deletions in ASD may be mostly a consequence of the alterations of the causative culprit genes for autism or genes responsible for mtDNA maintenance, or because of the harmful effect of environmental factors.

Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting.

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.

Genetic background of the hereditary spastic paraplegia phenotypes in Hungary - An analysis of 58 probands.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases with progressive lower limb spasticity and weakness. The aim of this study is to determine the frequency of different SPG mutations in Hungarian patients, and to provide further genotype-phenotype correlations for the known HSP causing genes. METHODS: We carried out genetic testing for 58 probands with clinical characteristics of HSP. For historical reasons, three different approaches were followed in different patients: 1) Sanger sequencing of ATL1 and SPAST genes, 2) whole exome, and 3) targeted panel sequencing by next generation sequencing. RESULTS: Genetic diagnosis was established for 20 probands (34.5%). We detected nine previously unreported mutations with high confidence for pathogenicity. The most frequently affected gene was SPAST with pathogenic or likely pathogenic mutations in 10 probands. The most frequently detected variant in our cohort was the SPG7 p.Leu78*, observed in four probands. Altogether five probands were diagnosed with SPG7. Additional mutations were detected in SPG11, ATL1, NIPA1, and ABCD1. CONCLUSION: This is the first comprehensive genetic epidemiological study of patients with HSP in Hungary. Next generation sequencing improved the yield of genetic diagnostics in this disease group even when the phenotype was atypical. However, considering the frequency of the HSP-causing gene defects, SPG4, the most common form of the disease, should be tested first to be cost effective in this economic region.