New Case of Spinocerebellar Ataxia, Autosomal Recessive 4, Due to VPS13D Variants.

Movement disorders such as bradykinesia, tremor, dystonia, chorea, and myoclonus most often arise in several neurodegenerative diseases with basal ganglia and white matter involvement. While the pathophysiology of these disorders remains incompletely understood, dysfunction of the basal ganglia and related brain regions is often implicated. The VPS13D gene, part of the VPS13 family, has emerged as a crucial player in neurological pathology, implicated in diverse phenotypes ranging from movement disorders to Leigh syndrome. We present a clinical case of VPS13D-associated disease with two variants in the VPS13D gene in an adult female. This case contributes to our evolving understanding of VPS13D-related diseases and underscores the importance of genetic screening in diagnosing and managing such conditions.

Leigh Syndrome: Spectrum of Molecular Defects and Clinical Features in Russia.

Leigh syndrome (LS), also known as infantile subacute necrotizing encephalopathy, is the most frequent mitochondrial disorder in children. Recently, more than 80 genes have been associated with LS, which greatly complicates the diagnosis. In this article, we present clinical and molecular findings of 219 patients with LS and give the detailed description of three cases with rare findings in nuclear genes MORC2, NARS2 and VPS13D, demonstrating wide genetic heterogeneity of this mitochondrial disease. The most common cause of LS in Russian patients are pathogenic variants in the SURF1 gene (44.3% of patients). The most frequent pathogenic variant is c.845_846delCT (66.0% of mutant alleles; 128/192), which is also widespread in Eastern Europe. Five main LS genes, SURF1, SCO2, MT-ATP6, MT-ND5 and PDHA1, account for 70% of all LS cases in the Russian Federation. Using next generation sequencing (NGS) technique, we were able to detect pathogenic variants in other nuclear genes: NDUFV1, NDUFS2, NDUFS8, NDUFAF5, NDUFAF6, NDUFA10, SUCLG1, GFM2, COX10, PMPCB, NARS2, PDHB and SLC19A3, including two genes previously associated with Leigh-like phenotypes-MORC2 and VPS13D. We found 49 previously undescribed nucleotide variants, including two deep intronic variants which affect splicing.

Plasma FGF-21 and GDF-15 are elevated in different inherited metabolic diseases and are not diagnostic for mitochondrial disorders.

Recently, the plasma cytokines FGF-21 and GDF-15 were described as cellular metabolic regulators. They share an endocrine function and are highly expressed in the liver under stress and during starvation. Several studies found that these markers have high sensitivity and specificity for the diagnosis of mitochondrial diseases, especially those with prominent muscular involvement. In our study, we aimed to determine whether these markers could help distinguish mitochondrial diseases from other groups of inherited diseases. We measured plasma FGF-21 and GDF-15 concentrations in 122 patients with genetically confirmed primary mitochondrial disease and 127 patients with non-mitochondrial inherited diseases. Although GDF-15 showed better analytical characteristics (sensitivity = 0.66, specificity = 0.64, area under the curve [AUC] = 0.88) compared to FGF-21 (sensitivity = 0.51, specificity = 0.76, AUC = 0.78) in the pediatric group of mitochondrial diseases, both markers were also elevated in a variety of non-mitochondrial diseases, especially those with liver involvement (Gaucher disease, galactosemia, glycogenosis types 1a, 1b, 9), organic acidurias and some leukodystrophies. Thus, the overall positive and negative predictive values were not acceptable for these measurements to be used as diagnostic tests for mitochondrial diseases (FGF-21 positive predictive value [PPV] = 34%, negative predictive value [NPV] = 73%; GDF-15 PPV = 47%, NPV = 28%). We suggest that FGF-21 and GDF-15 increase in patients with metabolic diseases with metabolic or oxidative stress and inflammation.

Identification of a Novel Indel Variant in the DARS2 Gene in Russian Patients with Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement. CASE PRESENTATION: Peripheral blood samples were collected from four patients from four unrelated families to extract genomic DNA. All patients underwent partial exon analysis of the DARS2 gene using Sanger sequencing, which detected the c.228-21_228-20delinsC variant in a heterozygous state. Further DNA from three patients was analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies, and one of the patients underwent whole genome sequencing. We identified a novel pathogenic variant c.1675-1256_*115delinsGCAACATTTCGGCAACATTCCAACC in the DARS2 gene. Three patients (patients 1, 2, and 4) had slowly progressive cerebellar ataxia, and two patients (patients 1 and 2) had spasticity. In addition, two patients (patients 2 and 4) showed signs of axonal neuropathy, such as decreased tendon reflexes and loss of distal sensitivity. Three patients (patients 1, 2, and 3) also had learning difficulties. It should be noted the persistent presence of characteristic changes in brain MRI in all patients, which emphasizes its importance as the main diagnostic tool for suspicion and subsequent confirmation of LBSL. Conclusions: We found a novel indel variant in the DARS2 gene in four patients with LBSL and described their clinical and genetic characteristics. These results expand the mutational spectrum of LBSL and aim to improve the laboratory diagnosis of this form of leukodystrophy.

Four Novel Disease-Causing Variants in the NOTCH3 Gene in Russian Patients with CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited disease with unknown mechanisms and a broad phenotypic spectrum. It is caused by pathogenic variants in the NOTCH3 gene. The symptoms of the disease mainly include recurrent strokes with vascular risk factors, migraine with aura, dementia, and mood disturbances. CASE PRESENTATION: Peripheral blood samples were collected from five patients from four unrelated families to extract genomic DNA. In four patients, analysis of exons 2, 3, 4, 5, 6 and adjacent intronic regions of the NOTCH3 gene was made via Sanger sequencing. Two previously undescribed nucleotide variants were identified in two patients: missense variant c.208G>T, (p.Gly70Cys) in exon 1 and splice-site variant c.341-1G>C in intron 3. Further DNA of two other patients were analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies. Two novel missense variants in the NOTCH3 gene were identified, c.1136G>A, (p.Cys379Tyr) in exon 7 and c.1547G>A, (p.Cys516Tyr) in exon 10. The pathogenic variant c.1547G>A, (p.Cys516Tyr) was confirmed in the fifth patient (family case) by Sanger sequencing. All patients had a history of headaches, transient ischemic attacks, memory impairment, and characteristics of MRI results. Three patients had strokes and two patients had psychiatric symptoms. CONCLUSION: We found four previously undescribed pathogenic variants in the NOTCH3 gene in five patients with CADASIL and described their clinical and genetic characteristics. These results expand the mutational spectrum of CADASIL.

New Acylcarnitine Ratio as a Reliable Indicator of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency.

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies are rare fatal disorders of fatty acid ß-oxidation with no apparent genotype-phenotype correlation. The measurement of acylcarnitines by MS/MS is a current diagnostic workup in these disorders. Nevertheless, false-positive and false-negative results have been reported, highlighting a necessity for more sensitive and specific biomarkers. This study included 54 patients with LCHAD/MTP deficiency that has been confirmed by biochemical and molecular methods. The analysis of acylcarnitines in dried blood spots was performed using ESI-MS/MS. The established "HADHA ratio" = (C16OH + C18OH + C18:1OH)/C0 was significantly elevated in all 54 affected individuals in comparison to the control group. Apart from 54 LCHAD deficiency patients, the "HADHA ratio" was calculated in 19 patients with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. As VLCAD-deficient patients did not show increased "HADHA ratio", the results emphasized the high specificity of this new ratio. Therefore, the "HADHA ratio" was shown to be instrumental in improving the overall performance of MS/MS-based analysis of acylcarnitine levels in the diagnostics of LCHAD/MTP deficiencies. The ratio was demonstrated to increase the sensitivity and specificity of this method and reduce the chances of false-negative results.

Expanding the genetic spectrum of the pyruvate carboxylase deficiency with novel missense, deep intronic and structural variants.

Introduction: Pathogenic variants in the pyruvate carboxylase (PC) gene cause a wide spectrum of recessive phenotypes, ranging from the early-onset fatal encephalopathy to the adult-onset benign form. Results: Patient 1 is a 6 y.o. boy with ataxia, hypoglycemia and episodes of lactic acidosis. WGS revealed the novel heterozygous missense variant c.1372A > G (p.Asn458Asp) in the PC gene. Additional analysis revealed discordant reads mapped to chromosomes 11 and 1, so a reciprocal translocation disrupted the PC gene was suspected. The translocation was validated via FISH-analysis and Sanger sequencing of its boundaries.Patient 2 is a 13 y.o. girl with psychomotor delay, episodes of lactic acidosis and ketonuria. WES revealed the novel homozygous intronic variant c.1983-116C > T. The PC's mRNA analysis demonstrated the exonization of several intron 16 sequences and some residual amount of WT mRNA isoform.Two other patients had more severe course of the disease. Their genotype represents missense variants in compound heterozygous and homozygous state (c.1876C > T (p.Arg626Trp), c.2606G > C (p.Gly869Ala), c.2435C > A (p.Ala812Asp). Conclusion: In patients with metabolic crises, lactic acidosis and hypoglycemia analysis of PC gene is recommended. WGS with deep bioinformatic analysis should be taken into consideration when none or the only one pathogenic variant in the PC gene is found.

POLG-associated ataxias can represent a substantial part of recessive and sporadic ataxias in adults.

OBJECTIVE: We aimed to analyze prevalence, clinical, and genetic characteristics of the POLG-associated ataxias in a cohort of recessive and sporadic ataxias in adults with previously excluded acquired ataxias. METHODS: We did a retrospective analysis of the medical records of 74 patients older than 18 years referred to the Research Center of Neurology between 2012 and 2019 with progressive sporadic or autosomal recessive ataxia with onset before 50 years of age. A stepwise approach in genetic testing was used. All patients with genetically confirmed POLG-associated disorders underwent clinical, biochemical, electrophysiological, and neuroimaging assessments. RESULTS: In our cohort of 74 adult patients with autosomal recessive and sporadic ataxias, POLG-related disease was identified in 11 individuals (14.9 %). The median age of onset was 30 years. One patient had a positive family history. The core clinical syndrome included external ophthalmoparesis, cerebellar signs, and sensory neuropathy. In all patients, the Montreal Cognitive Assessment score was less than 26. All but 3 patients had specific brain MRI changes. Mutation spectrum of the POLG gene in our cohort is discussed. CONCLUSION: Our study shows that POLG-associated ataxias comprise a significant part of the recessive and sporadic ataxias in adults in the Russian population after excluding acquired causes of ataxic disorders. We suggest first screening patients with specific clinical and (or) neuroimaging features for the population-specific common POLG mutations, followed by the NGS panel testing where necessary. In future clinical studies, thorough cognitive and neuropsychiatric profiling is needed to complete the phenotype of the POLG-related disorders.

Three rare pathogenic mtDNA substitutions in LHON patients with low heteroplasmy.

In this article we present clinical, molecular and biochemical investigations of three patients with LHON caused by rare point substitutions in mtDNA. One patient harbours the known mtDNA mutation (m.13513 G>A), the others have new variants (m.13379 A>G in MT-ND5 gene and m.14597 A>G in MT-ND6 gene, which has never been previously associated with LHON). NGS analysis of a whole mtDNA derived from patient's blood revealed a low mutation load (24%, 47%, 23% respectively). Our data, including family segregation analysis, measurement of reactive oxygen species (ROS) production and cytotoxic effect of paraquat and high-resolution respirometry, showed that nucleotide variant m.14597 A>G can be classified as pathogenic mutation.