Objective: Our aim is to offer a complete and reliable diagnostic service excluding false negative.

Background: Heterozygous variants in the GBA gene are the most common genetic risk factors for Parkinson’s disease (PD) worldwide [1]. Molecular analysis of the GBA gene is complicated by the presence of a highly homologous pseudogene (GBAP1). The coding region of the GBA gene shares 96% homology with the exonic region of GBAP1 but the sequence homology increases up to 98% in the region between intron 8 and the 3′ untranslated region (UTR) [2]. The high homology and closeness between GBA and GBAP1 cause homologous recombination events such as crossover, mainly at the end of the gene where the homology is highest. Since 2015, a genetic panel for the screening of all known genes associated with Parkinson’s disease has been available in the Genetic Movement Disorders Laboratory of the Neurological Institute “Carlo Besta” in Milan. Over the years we have analysed 314 samples, 41 of these were positive (13.1%). Since NGS technology cannot detect the presence of recombinant mutations in the GBA gene, we decided to reanalyse samples tested negative by custom panel analysis (279 samples) using a low-cost and rapid technique.

Method: We developed a multiplex allele-specific PCR (AS-PCR) [3] which simultaneously amplifies a specific portion of GBA from exon 8 to exon 11. The allele-specific oligonucleotides, used in this multiplex PCR, allow the discrimination of GBA versus GBAP1. AS-PCR is able to detect four missense substitutions (p.L444P; p.N370S; p.T369M; p.E326K), that together account for 80% of early-onset PD-associated GBA mutations and in addition the two complex alleles [4], named RecNcil and Recdelta55, that arise from the presence of 2 or more cis variants resulting from a rearrangement with consequent insertion of part of GBAP1 in GBA [5].

Results: The analysis identified 5 positive samples (1.8% this value is in line with what has been published in the literature so far [3]): 4 samples carrying the complex allele RecNcil and 1 sample carrying the single missense mutation p.T369M, unexpectedly not identified with NGS. All samples were reanalysed and confirmed with Sanger sequencing method.

Conclusion: We confirmed that the use of multiplex AS-PCR analysis for the GBA gene is a sensitive technique for the diagnosis of GBA-related PD and should be complemented with custom panel analysis in NGS.

References: 1- Sidransky E, Nalls MA, Aasly JO, Aharon-Peretz J, Annesi G, Barbosa ER, Bar-Shira A, Berg D, Bras J, Brice A, Chen CM, Clark LN, Condroyer C, De Marco EV, Dürr A, Eblan MJ, Fahn S, Farrer MJ, Fung HC, Gan-Or Z, Gasser T, Gershoni-Baruch R, Giladi N, Griffith A, Gurevich T, Januario C, Kropp P, Lang AE, Lee-Chen GJ, Lesage S, Marder K, Mata IF, Mirelman A, Mitsui J, Mizuta I, Nicoletti G, Oliveira C, Ottman R, Orr-Urtreger A, Pereira LV, Quattrone A, Rogaeva E, Rolfs A, Rosenbaum H, Rozenberg R, Samii A, Samaddar T, Schulte C, Sharma M, Singleton A, Spitz M, Tan EK, Tayebi N, Toda T, Troiano AR, Tsuji S, Wittstock M, Wolfsberg TG, Wu YR, Zabetian CP, Zhao Y, Ziegler SG. Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N Engl J Med. 2009 Oct 22;361(17):1651-61. doi: 10.1056/NEJMoa0901281. PMID: 19846850; PMCID: PMC2856322.
2- Zampieri S, Cattarossi S, Bembi B, Dardis A. GBA Analysis in Next-Generation Era: Pitfalls, Challenges, and Possible Solutions. J Mol Diagn. 2017 Sep;19(5):733-741. doi: 10.1016/j.jmoldx.2017.05.005. Epub 2017 Jul 18. PMID: 28727984.
3- Straniero L, Rimoldi V, Melistaccio G, Di Fonzo A, Pezzoli G, Duga S, Asselta R. A rapid and low-cost test for screening the most common Parkinson’s disease-related GBA variants. Parkinsonism Relat Disord. 2020 Nov;80:138-141. doi: 10.1016/j.parkreldis.2020.09.036. Epub 2020 Sep 22. PMID: 32987361.
4- Straniero L, Asselta R, Bonvegna S, Rimoldi V, Melistaccio G, Soldà G, Aureli M, Della Porta M, Lucca U, Di Fonzo A, Zecchinelli A, Pezzoli G, Cilia R, Duga S. The SPID-GBA study: Sex distribution, Penetrance, Incidence, and Dementia in GBA-PD. Neurol Genet. 2020 Oct 20;6(6):e523. doi: 10.1212/NXG.0000000000000523. PMID: 33209983; PMCID: PMC7670574.
5- Petrucci S, Ginevrino M, Trezzi I, Monfrini E, Ricciardi L, Albanese A, Avenali M, Barone P, Bentivoglio AR, Bonifati V, Bove F, Bonanni L, Brusa L, Cereda C, Cossu G, Criscuolo C, Dati G, De Rosa A, Eleopra R, Fabbrini G, Fadda L, Garbellini M, Minafra B, Onofrj M, Pacchetti C, Palmieri I, Pellecchia MT, Petracca M, Picillo M, Pisani A, Vallelunga A, Zangaglia R, Di Fonzo A, Morgante F, Valente EM; ITA-GENE-PD Study Group. GBA-Related Parkinson’s Disease: Dissection of Genotype-Phenotype Correlates in a Large Italian Cohort. Mov Disord. 2020 Nov;35(11):2106-2111. doi: 10.1002/mds.28195. Epub 2020 Jul 13. PMID: 32658388.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/the-development-of-a-multiplex-allele-specific-pcr-to-improve-the-diagnostic-yield-of-ngs-in-the-gba-gene-screening/