Background: Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is clinically characterized by the presence of motor (bradykinesia, rigidity, rest tremor and postural instability) and non-motor symptoms (cognitive impairment, autonomic dysfunction, sleep disorders, depression and hyposmia). The aetiology of PD is unknown except for a small but significant contribution of monogenic forms. Sources of data: No new data were generated or analyzed in support of this review. Areas of agreement: Up to 15% of PD patients carry pathogenic variants in PD-associated genes. Some of these genes are associated with mendelian inheritance, while others act as risk factors. Genetic background influences age of onset, disease course, prognosis and therapeutic response. Areas of controversy: Genetic testing is not routinely offered in the clinical setting, but it may have relevant implications, especially in terms of prognosis, response to therapies and inclusion in clinical trials. Widely adopted clinical guidelines on genetic testing are still lacking and open to debate. Some new genetic associations are still awaiting confirmation, and selecting the appropriate genes to be included in diagnostic panels represents a difficult task. Finally, it is still under study whether (and to which degree) specific genetic forms may influence the outcome of PD therapies. Growing points: Polygenic Risk Scores (PRS) may represent a useful tool to genetically stratify the population in terms of disease risk, prognosis and therapeutic outcomes. Areas timely for developing research: The application of PRS and integrated multi-omics in PD promises to improve the personalized care of patients.

Background and Objective: Early‐onset Parkinson's disease (EOPD) commonly recognizes a genetic basis; thus, patients with EOPD are often addressed to diagnostic testing based on next‐generation sequencing (NGS) of PD‐associated multigene panels. However, NGS interpretation can be challenging in a diagnostic setting, and few studies have addressed this issue so far.Methods: We retrospectively collected data from 648 patients with PD with age at onset younger than 55 years who underwent NGS of a minimal shared panel of 15 PD‐related genes, as well as PD‐multiplex ligation‐dependent probe amplification in eight Italian diagnostic laboratories. Data included a minimal clinical dataset, the complete list of variants included in the diagnostic report, and final interpretation (positive/negative/inconclusive). Patients were further stratified based on age at onset ≤40 years (very EOPD, n = 157). All variants were reclassified according to the latest American College of Medical Genetics and Genomics criteria. For classification purposes, PD‐associated GBA1 variants were considered diagnostic.Results: In 186 of 648 (29%) patients, the diagnostic report listed at least one variant, and the outcome was considered diagnostic (positive) in 105 (16%). After reanalysis, diagnosis changed in 18 of 186 (10%) patients, with 5 shifting from inconclusive to positive and 13 former positive being reclassified as inconclusive. A definite diagnosis was eventually reached in 97 (15%) patients, of whom the majority carried GBA1 variants or, less frequently, biallelic PRKN variants. In 89 (14%) cases, the genetic report was inconclusive.Conclusions: This study attempts to harmonize reporting of PD genetic testing across several diagnostic labs and highlights current difficulties in interpreting genetic variants emerging from NGS‐multigene panels, with relevant implications for counseling. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

GBA1 variants are the most common genetic risk factor for Parkinson disease (PD). Sequencing of GBA1 on a large scale represents a burdensome task with currently adopted diagnostic techniques, namely Sanger sequencing and conventional short read next generation sequencing (sr-NGS). The high degree of sequence homology between GBA1 and its pseudogene GBA1LP is the major driver behind this complexity, leading to false positive and false negative results. We designed, optimized and validated LONG-NEXT, a new NGS-based strategy to streamline large scale GBA1 sequencing. LONG-NEXT relies on a specific long-range PCR, encompassing the whole GBA1 gene, in one fragment (6.5 kb), followed by short-read NGS and a tailored bioinformatic pipeline masking the GBA1LP sequence on the reference genome. This protocol was optimized and tested on selected cases suspected of diagnostic mistakes by conventional testing (n = 13) and then validated on consecutively collected PD patients already screened either by Sanger sequencing (n = 101) or conventional sr-NGS (n = 294). LONG-NEXT reanalysis of 13 patients disclosed: 3 false positive cases due to mismapping of pseudogene reads on GBA1, 4 false homozygotes due to PCR-related allele dropout events, and 6 false negative cases, due to misalignment of GBA1 reads against the pseudogene or PCR-related allele dropout events. The validation phase disclosed one additional false homozygote in the Sanger cohort, and one false negative result in the sr-NGS cohort. LONG-NEXT is a reliable, fast, cost-effective alternative for GBA1 sequencing and may prove strategic in light of current genotype-based tailored therapies specifically targeting GBA1-PD patients.

Background and Aims: POLR3B gene encodes a subunit of RNA polymerase III (Pol III). Biallelic mutations in POLR3B are associated with leukodystrophies, but recently de novo heterozygous mutations have been described in early onset peripheral demyelinating neuropathies with or without central involvement. Here, we report the first Italian case carrying a de novo variant in POLR3B with a pure neuropathy phenotype and primary axonal involvement of the largest nerve fibers.Methods: Nerve conduction studies, sympathetic skin response, dynamic sweat test, tactile and thermal quantitative sensory testing and brain magnetic resonance imaging were performed according to standard procedures. Histopathological examination was performed on skin and sural nerve biopsies. Molecular analysis of the proband and his relatives was performed with Next Generation Sequencing. The impact of the identified variant on the overall protein structure was evaluated through rotamers method.Results: Since his early adolescence, the patient presented with signs of polyneuropathy with severe distal weakness, atrophy, and reduced sensation. Neurophysiological studies showed a sensory‐motor axonal polyneuropathy, with confirmed small fiber involvement. In addition, skin biopsy and sural nerve biopsy showed predominant large fibers involvement. A trio's whole exome sequencing revealed a novel de novo variant p.(Arg1046Cys) in POLR3B, which was classified as Probably Pathogenic. Molecular modeling data confirmed a deleterious effect of the variant on protein structure.Interpretation: Neurophysiological and morphological findings suggest a primary axonal involvement of the largest nerve fibers in POLR3B‐related neuropathies. A partial loss of function mechanism is proposed for both neuropathy and leukodystrophy phenotypes.

Background The application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The objective is to reduce the number of variants, focusing only on those with pathogenic significance. The 2015 American College of Medical Genetics and Genomics (ACMG) guidelines aid in achieving this goal, but it is now evident that a pathology or gene-specific review of these rules is essential to avoid misinterpretations that may result from blindly applying the criteria. This study demonstrates how revised ACMG criteria, combined with CMT-specific literature data and expertise, can alter the final classification of a variant. Methods We reviewed ACMG criteria based on current knowledge of CMT and provided suggestions for adapting them to the specificities of CMT. Results Of the 226 index patients analysed, a diagnostic yield of 20% was obtained. It is worth noting that the 9% of cases had their final diagnosis changed with the application of the revised criteria, often resulting in the loss of the pathogenic classification of a variant. Conclusions The widespread availability of high-throughput sequencing technologies has enabled genetic testing even for laboratories without specific disease expertise. Disease-specific ACMG criteria can be a valuable tool to prevent the proliferation of variants of uncertain significance and the misinterpretation of variants.