Mutations, Genes, and Phenotypes Related to Movement Disorders and Ataxias

Citation: Martínez-Rubio, D.; Hinarejos, I.; Sancho, P.; Gorría-Redondo, N.; Bernadó-Fonz, R.; Tello, C.; Marco-Marín, C.; Martí-Carrera, I.; Martínez-González, M.J.; García-Ribes, A.; et al. Mutations, Genes, and Phenotypes Related to Movement Disorders and Ataxias. Int. J. Mol. Sci. 2022,23, 11847. https://doi.org/10.3390/ ijms231911847 Academic Editors: Orietta Pansarasa and Matteo Bordoni Received: 5 September 2022 Accepted: 3 October 2022 Published: 6 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Mutations, Genes, and Phenotypes Related to Movement Disorders and Ataxias Dolores Martínez-Rubio 1,2,†, Isabel Hinarejos 1,2,† , Paula Sancho 1,†, Nerea Gorría-Redondo 3, Raquel Bernadó-Fonz 3, Cristina Tello 1, Clara Marco-Marín4, Itxaso Martí-Carrera 5, María Jesús Martínez-González 6, Ainhoa García-Ribes 6, Raquel Baviera-Muñoz 2,7,8 , Isabel Sastre-Bataller 2,8, Irene Martínez-Torres 2,8, Anna Duat-Rodríguez 9, Patrícia Janeiro 10, Esther Moreno 11, Leticia Pías-Peleteiro 12, Mar O’Callaghan Gordo 12,Ángeles Ruiz-Gómez 13, Esteban Muñoz 14 , Maria Josep Martí14, Ana Sánchez-Monteagudo 1,2, Candela Fuster 1, Amparo Andrés-Bordería1, Roser Maria Pons 15, Silvia Jesús-Maestre 16, Pablo Mir 16, Vincenzo Lupo 1, Belén Pérez-Dueñas 17, Alejandra Darling 12, Sergio Aguilera-Albesa 3,*,† and Carmen Espinós1,2,18,*,† 1Rare Neurodegenerative Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), 46012 Valencia, Spain 2Joint Unit CIPF-IIS La Fe Rare Diseases, 46012 Valencia, Spain 3Paediatric Neurology Unit, Department of Paediatrics, Hospital Universitario de Navarra, Navarrabiomed, 31008 Pamplona, Spain 4Structural Enzymopathology Unit, Instituto de Biomedicina de Valencia (IBV), Consejo Superior de Investigaciones Científicas (CSIC), Centro de Investigación Biomédica de Enfermedades Raras (CIBERER-ISCIII), 46010 Valencia, Spain 5Biodonostia Health Research Institute, Paediatric Group, Donostia University Hospital, Department of Paediatrics, University of the Basque Country UPV/EHU, 20014 San Sebastian, Spain 6Paediatric Neurology Unit, Hospital Universitario Cruces, 48903 Barakaldo, País Vasco, Spain 7Health Research Institute, Hospital Universitari i Politècnic La Fe, 46026 Valencia, Spain 8Movement Disorders Unit, Neurology Department, Hospital Universitari i Politècnic La Fe, 46026 Valencia, Spain 9Paediatric Neurology Unit, Hospital Infantil Universitario Niño Jesús, 28009 Madrid, Spain 10 Centro de Referencia de Doenças Hereditarias do Metabolismo, CHULN, Hospital Santa Maria, 1649-035 Lisbon, Portugal 11 Department of Paediatrics, Hospital Regional Universitario, 29010 Malaga, Spain 12 Paediatric Neurology Unit, Hospital Sant Joan de Déu, 08950 Barcelona, Spain 13 Department of Paediatrics, Hospital Universitari Son Espases, 07120 Palma de Mallorca, Spain 14 Unit of Parkinson and Movement Disorders, Department of Neurology, Hospital Universitari Clínic, 08036 Barcelona, Spain 15 Paediatric Neurology, Hospital Agia Sofía, 11527 Athens, Greece 16 Movement Disorders Unit, Department of Neurology and Clinical Neurophysiology, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED-ISCIII), 41013 Seville, Spain 17 Department of Paediatric Neurology, Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca, 08035 Barcelona, Spain 18 Department of Biotechnology, Faculty of Veterinary and Experimental Sciences, Universidad Católica de Valencia “San Vicente Mártir”, 46001 Valencia, Spain *Correspondence: [email protected] (S.A.-A.); [email protected] (C.E.); Tel.: +34-848-422-563 (S.A.-A.); +34-963-289-680 (C.E.) † These authors contributed equally to the manuscript. Abstract: Our clinical series comprises 124 patients with movement disorders (MDs) and/or ataxia with cerebellar atrophy (CA), many of them showing signs of neurodegeneration with brain iron accumulation (NBIA). Ten NBIA genes are accepted, although isolated cases compatible with abnormal brain iron deposits are known. The patients were evaluated using standardised clinical assessments of ataxia and MDs. First, NBIA genes were analysed by Sanger sequencing and 59 patients achieved a diagnosis, including the detection of the founder mutation PANK2 p.T528M in Romani people. Then, we used a custom panel MovDisord and/or exome sequencing; 29 cases were solved with a great genetic heterogeneity (34 different mutations in 23 genes). Three patients presented brain iron deposits with Fe-sensitive MRI sequences and mutations in FBXO7,GLB1, and KIF1A, suggesting Int. J. Mol. Sci. 2022,23, 11847. https://doi.org/10.3390/ijms231911847 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 11847 2 of 26 an NBIA-like phenotype. Eleven patients showed very early-onset ataxia and CA with cortical hyperintensities caused by mutations in ITPR1,KIF1A,SPTBN2,PLA2G6, PMPCA, and PRDX3. The novel variants were investigated by structural modelling, luciferase analysis, transcript/minigenes studies, or immunofluorescence assays. Our findings expand the phenotypes and the genetics of MDs and ataxias with early-onset CA and cortical hyperintensities and highlight that the abnormal brain iron accumulation or early cerebellar gliosis may resembling an NBIA phenotype. Keywords: movement disorders; ataxia; cerebellar atrophy; neurodegeneration with brain iron accumulation (NBIA); gene panel; exome sequencing 1. Introduction In 2015, we initiated a new research line focused on disorders associated with neurodegeneration with brain iron accumulation (NBIA). This group of syndromes is characterised by movement disorders (MDs) with the common feature of abnormal deposits of iron in the brain, although clinical and neuroimaging features are variable, reflecting the genetic heterogeneity in this group [ 1 – 3 ]. Pantothenate kinase-associated neurodegeneration (PKAN), caused by PANK2 mutations [ 4 ], is the most frequent NBIA phenotype; dystonia, parkinsonism, and cognitive decline over the years are core features. In PLA2G6-associated neurodegeneration (PLAN), the second most frequent NBIA, hypotonia, ataxia, and early progressive cerebellar atrophy (CA) are the hallmark of the disease, with brain iron deposits detected in only 50% or less of cases [ 5 ]. Different clinical characteristics are predominant in other NBIA disorders, as ataxia, spastic paraparesis, peripheral neuropathy, optic atrophy, and neuropsychiatric problems. Thus, the clinical phenotype and brain magnetic resonance imaging (MRI) patterns evolve differently in each disorder and depend on the stage of the disease and the age of the patient [3]. In clinical practice, genetic diagnosis in these phenotypes may end up being a cumbersome odyssey, due to the difficulty in making reliable genotype-phenotype correlations. Assessment of next generation sequencing (NGS) tools has been reported for cohorts with MDs [ 6 ], and more frequently for specific sets of entities such as ataxia [ 7 , 8 ] or hereditary spastic paraplegia (HSP) [ 9 , 10 ]. Targeted gene sequencing likely represents the most costeffective option, whereas the gold standard seems to be whole exome sequencing (WES) as it covers the known coding genome. In our cohort, the sequencing of selected NBIA genes in 114 patients provided the genetic diagnosis of 59 patients, including the detection of the founder mutation PANK2 p.T528M in the Roma population. We then studied the undiagnosed patients using a custom panel MovDisord and WES-trio, plus 10 additional cases investigated exclusively by WES-proband. Twenty-nine probands achieved a conclusive molecular diagnosis with a great genetic heterogeneity (34 different disease-causing mutations in 23 genes). Considering the whole series, the diagnostic success rate was 70.97%. We expanded MDs phenotypes with brain iron accumulation, but also ataxia phenotypes with CA and cortical hyperintensities, from classical PLAN to non-progressive congenital ataxia (NPCA) phenotypes. 2. Results 2.1. Genetics The mutational screening of the most frequent NBIA genes revealed that 41 (35.96%) and 14 (12.28%) out of 114 patients carried biallelic mutations in PANK2 or PLA2G6, respectively (Figure 1). Regarding other NBIA genes, one proband harboured a causative variant in WDR45 (c.182A>C, p.N61T); two in FTL (c.286G>A, p.A96T; c.509_*4del, p.L170_Pfs*8); and another one in CP (c.1652C>T, p.T551I/c.2684G>C, p.G895A). Taken together, the etiological diagnosis was attained in 59 (51.75%) patients. The NBIA mutations detected in our series are available at https://espinos.cipf.es (accessed on 1 August 2022). Int. J. Mol. Sci. 2022,23, 11847 3 of 26 Figure 1. Casuistry and strategy. ( A ) Pipeline designed for genetic analysis of a cohort of patients with movement disorders and ataxia, which includes different approaches from candidate gene to gene panel and WES (whole exome sequencing) carried out in a cohort of 124 probands. ( B ) Distribution of cases studied by gene panel MovDisord. In 59 probands, a definitive diagnosis was achieved by Sanger sequencing and MLPA (in blue): 41 patients carried mutations in PANK2, 14 in PLA2G6, and 4 in other NBIA genes (http://espinos.cipf.es/index.php/en/mutations-db, accessed on 1 August 2022). Using a custom panel MovDisord, the remaining 55 probands were investigated and the causative mutations were identified in 19 cases (in orange). Finally, nine patients were further studied by WES and in three cases the clinical variant was detected (in green). To compare both approaches (gene panel versus WES), ten patients were investigated by WES only, and the disease-causing mutations were detected in seven of them (in purple). NBIA: neurodegeneration with brain iron accumulation. MD: Movement disorders. MLPA: multiplex ligation-dependent probe amplification. The seven unrelated Roma patients from Spain (homozygous for the PANK2 c.1583C>T, p.T528M mutation), shared the same haplotype [A-268-C-T-C-G-285] that stretched from rs1078152 to D20S116, encompassing a 0.9 Mb region (Table S1). The Hungarian Roma proband had a narrower haplotype for both alleles, which included the intragenic markers (13.1 Kb) only. Two Greek heterozygous carriers of the PANK2 c.1583C>T mutation presented the same haplotype as the Spanish patients, and one Greek proband showed a finer haplotype from rs1078152 to rs7270329 (0.8 Mb). Lastly, the non-Roma Spanish proband heterozygous for the investigated variant had a different haplotype, suggesting different mutational events for PANK2 c.1583C>T. The 55 unsolved patients were investigated by a custom panel MovDisord (Figure 1). The mean coverage among all samples was >400 × , the lowest mean coverage achieved was 233 × , and the highest 605 × (Figure S1). In all cases, at least 99.7% of the bases were covered with >20 reads and mean gene coverage was >65 × (Table S2). Panel MovDisord was robust in terms of coverage, as well as read depth for all genes and samples. Nineteen out of fifty-five patients were genetically diagnosed (Table 1and Figure 1). Nine unsolved patients were then investigated by WES-trio and mutations in PLEKHG2, NR4A2, and PRDX3 were identified in three probands (Table 1). None of these three genes were included in the panel. In parallel, seven out of ten patients, investigated by WES-proband only, obtained a conclusive genetic diagnosis (Table 1and Figure 1). It is worth mentioning that the MD-319 case (LRRK2 gene) was cleared up after a reanalysis of WES data two years after the initial analysis. In total, 29 patients were diagnosed. Int. J. Mol. Sci. 2022,23, 11847 4 of 26 Table 1. Genetic findings of the 29 solved cases. Patient Gene RefSeq Position DNA Change Protein Change Prediction ♦rs number (MAF) ‡References PMID §Method MD-208 CASK NM_003688.3 X:41383202 c.2589+2T>G — P NA None Gene Panel MD-216 CPLANE1 NM_023073.3 5:37201821 c.3379T>G p.S1127A LP rs776423792 (1.193 ×10−5)None Gene Panel 5:37164372 c.7588+3A>G — P NA None MD-122 EXOSC3 NM_013042.3 9:37783990 c.395A>C p.D132A P rs141138948 (4.067 ×10−4) 22544365, 23564332, 23564332, 23975261, 24524299, 25533962, 27777260, 28687512, 30950035, 31692161 Gene Panel HOMOZYGOSIS (No Consanguinity) MD-012 EXOSC3 NM_013042.3 9:37783990 c.395A>C p.D132A P rs141138948 (4.067 ×10−4) 22544365, 23564332, 23564332, 23975261, 24524299, 25533962, 27777260, 28687512, 30950035, 31692161 Gene Panel HOMOZYGOSIS (No Consanguinity) MD-018 #FBXO7 NM_012179.3 22:32875213 c.368C>G p.S123 * P NA 32767480 Gene Panel HOMOZYGOSIS (Consanguinity) MD-137 FUCA1 NM_000147.4 1:24194637 c.140G>C p.R47P LP NA None Gene Panel HOMOZYGOSIS (Consanguinity) MD-020 GLB1 NM_000404.3 3:33114105 c.176G>A p.R59H P rs72555392 (3.607 ×10−5)10338095, 17664528, 28939701, 31761138 Gene Panel 3:33114174 c.107A>G p.Y36C P rs748345527 (4.809 ×10−5)None MD-270 HEXA NM_000520.5 15:72646027 c.459+5G>A — P rs762060470 (2.829 ×10−5)1837283, 25525159 Gene Panel 15:72638893 c.1305C>T — P rs587779406 (8.488 ×10−5)20363167, 25606403 Int. J. Mol. Sci. 2022,23, 11847 5 of 26 Table 1. Cont. Patient Gene RefSeq Position DNA Change Protein Change Prediction ♦rs number (MAF) ‡References PMID §Method MD-320 ITPR1 NM_00116872.1 3:4687357 c.800C>T p.T267M P rs797044955 (NA) 24091540, 28659154, 29878067, 29925855, 29925855, 30842224, 31632679, 32695065 WES-proband MD-106 ITPR1 NM_00116872.1 3:4687362 c.805C>T p.R269W P NA 27062503, 25533962, 28826917, 28191890, 28135719, 28659154, 29925855 Gene Panel MD-041 ITPR1 NM_00116872.1 3:4706967 c.1655A>G p.Y552C LP NA None Gene Panel MD-200 KIF1A NM_001244008.1 2:241725854 c.506C>G p.R169T LP NA 34121983 Gene Panel MD-189 KIF1A NM_001244008.1 2:241715280 c.946C>T p.R316W P NA 25265257, 26354034, 28554332 Gene Panel MD-299 KIF1A NM_001244008.1 2:241715280 c.946C>T p.R316W P NA 25265257, 26354034, 28554332 WES-proband MD-319 LRRK2 NM_198578.4 12:40734202 c.6055G>A p.G2019S P rs34637584 (4.884 ×10−4) 15726496, 16102999, 16333314, 16750377, 16966501, 17060589, 17116211, 17210620, 17200152, 19072560, 20008657, 19283415, 21686713, 19302196, 19741132 σ WES-proband HOMOZYGOSIS (Consanguinity) MD-277 #NR4A2 NM_006186.4 2:157184954 c.956G>A p.R319Q LP NA 33585677 WES-trio MD-173 PDGFB NM_002608.3 22:39621853 c.602-1G>C — P NA None Gene Panel MD-252 PNKD NM_015488.4 2:219187987 c.-4C>G — LP rs1461115674 (8.152 ×10−6)None Gene Panel MD-341 PLA2G6 NM_003560 22:38519251 c.1442T>A p.L481Q LP rs587784330 (5.922 ×10−6)16783378, 24870368, 25164370 WES-proband HOMOZYGOSIS (Consanguinity) MD-181 PLEKHG2 NM_022835.2 19:39905680 c.158C>T p.T53I P NA None WES-trio HOMOZYGOSIS (Consanguinity) MD-323 PMPCA NM_015160.2 9:139310844 c.633+1G>A — P rs1442110087 (7.961 ×10−6)None WES-proband 9:139306513 c.136T>C p.S46P LP NA None Int. J. Mol. Sci. 2022,23, 11847 6 of 26 Table 1. Cont. Patient Gene RefSeq Position DNA Change Protein Change Prediction ♦rs number (MAF) ‡References PMID §Method MD-174 #PRDX3 NM_006793.5 10:120931956 c.489C>G p.R163E P NA 35766882 WES-trio HOMOZYGOSIS (Consanguinity) MD-296 QARS1 NM_005051 3:49138870 c.794G>A p.R265H LP rs916890735 (6.575 ×10−6)None WES-proband HOMOZYGOSIS (Consanguinity) MD-126 #REEP1 arr[hg19]2p11.2 (chr2:83,335,425-87,271,924; Hg 19) P — 22062632, 24986827 Gene Panel MD-307 RPGRIP1L NM_015272 16:53720424 c.697A>T p.K233* P rs121918197 (2.788 ×10−5)17558409, 25525159 WES-proband 16:53686828 c.1769_1770delCT p.S590Cfs* P NA None MD-159 #SPG7 NM_003119.3 16:89613145 c.1529C>T p.A510V LP rs61755320 (2.899 ×10−5) 18799786, 20981092, 20186691, 21623769, 22995991, 23269439, 22571692, 25133958, 25525159, 26626314, 29057857, 27957547, 29026558, 28362824, 28832565, 29482223, 29913018, 30369941, 31433872, 30098094, 31316545, 29915382, 30537300, 31692161 Gene Panel 16:89616953 c.1715C>T p.A572V P rs72547551 (3.537 ×10−5)14985266, 25681447, 29482223 MD-219 #SPTBN2 NM_006946.3 11:66483417 c.193A>G p.K65E P NA 33801522 Gene Panel MD-207 #SPTBN2 NM_006946.3 11:66481110 c.764A>G p.D255G P NA 33801522 Gene Panel MD-153 TPP1 NM_000391.3 11:6636487 c.1340G>A p.R447H P rs119455956 (7.953 ×10−5)10330339, 19038966, 20340139, 26143525, 29655203 Gene Panel 11:6640007 c.229G>C p.G77R P rs121908195 (7.969 ×10−5)26633542, 31741823 AD, Autosomal Dominant; AR, Autosomal Recessive; LP, Likely Pathogenic; NA, Not-Available; P, Pathogenic; PMID, PubMed Identifier; RefSeq, Reference Sequence; SCA, Spinocerebellar Ataxia; SPG: Spastic Paraplegia; WES, Whole Exome Sequencing; XLD, X-linked dominant. ♦ Prediction of pathogenicity performed according to the ACMG/AMP guidelines [ 11 ]. ‡ Consulted database gnomAD v2.1.1, except for rs916890735 that is only annotated in gnomAD v3.1.1 (accessed on 1 August 2022). § References associated with each mutation in the Human Mutation Database (HGMD ® ) Professional 2022.2 (Qiagen, Santa Clarita, CA, USA; accessed om 1 August 2022). σ The LRRK2 p.G2019S change is reported in at least 119 articles. #Clinical features of patients MD-018, MD-277, MD-174, MD-126, MD-159, MD-219, and MD-207 were previously reported [12–17]. Int. J. Mol. Sci. 2022,23, 11847 7 of 26 Two cases were familial and one case currently sporadic (MD-252), likely would be a familial one (the daughter’s proband is asymptomatic; Figure S2 and Table 2); twentyfive cases were sporadic (11 de novo cases); and one case remained unclassified due to lack of information. Most of the deleterious changes were transmitted in an AR fashion (16 cases) or in an AD manner (12 cases), and only one case presented an X-linked dominant (XLD) inheritance. Nine AR cases had mutations in homozygosis, and in seven of them, the parents were consanguineous (five patients from Morocco, one from Greece, and one from Spain). Two mutations were identified twice: EXOSC3 p.D132A in homozygosis in non-consanguineous cases, and KIF1A p.R316W with a de novo presentation (Tables 1and 2). As a whole, 34 mutations were detected, 17 of them not previously associated with disease, in 23 different genes, including those formerly published [ 12 – 17 ]. Int. J. Mol. Sci. 2022,23, 11847 8 of 26 Table 2. Clinical features of the 29 solved cases. Patient Sex Gene Origin Presentation Inheritance Disease (OMIM) Age of Onset Age at Testing Early Symptoms Brain MRI Additional Clinical Features MD-208 Female CASK Spain de novo XLD Mental retardation and microcephaly with pontine and cerebellar hypoplasia (300749) 1 mo 3 yo Hypotonia Microcephaly DD Severe PCH Mild limb dystonia, choreoathetosis, progressive scoliosis from 3 yo MD-216 Male CPLANE1 Morocco Familial AR Joubert syndrome 17 (614615) 1 yo 10 yo DD Hypotonia, ataxia OMA Molar tooth sign Cerebellar hypoplasia ID, ataxia and OMA improve with age MD-122 Female EXOSC3 Spain Sporadic AR Pontocerebellar hypoplasia type 1B (614678) 3 mo 3 yo Hypotonia, axial hyperextension, strabismus Progressive vermis atrophy Spasticity progressing to flaccid paralysis from 3-yo, g-tube feeding, neurogenic EMG pattern, exitus at 4 yo MD-012 Male EXOSC3 Spain Sporadic AR 4 mo 8 yo Hypotonia, DD Progressive vermis atrophy Spasticity from 4 yo, axonal neuropathy with loss of tendon reflexes from 5 yo, g-tube feeding, limb dystonia from 6 yo MD-018 # Female FBXO7 Morocco Sporadic AR Parkinson disease 15 (260300) 2 yo 21 yo Mild DD, infection-triggered acute ataxia at 2 yo CA from 10 yo Brain iron deposits from 15 yo Absence epilepsy from 5 yo, acute encephalopathy at 12 yo, progressive deterioration, spastic paraparesis, drug-resistant seizures, optic neuropathy, parkinsonism from 15 yo MD-137 Male FUCA1 Greece Sporadic AR Fucosidosis (230000) 3 yo 10 yo Speech difficulties, gait disturbances GP T2 hypointensity from 10 yo Static encephalopathy, progression of gait disorder with dystonia, ID, dysmorphic features, ADHD symptoms, motor stereotypes MD-020 Female GLB1 Spain Sporadic AR GM1-gangliosidosis (230500, 230600, 230650) 3 yo 12 yo Speech difficulties, motor deterioration Brain iron deposits from 10 yo Oromandibular dystonia, dysarthria, neuropathy, spasticity, scoliosis MD-270 Female HEXA Spain NA AR GM2-gangliosidosis (272800) 26 yo 23 yo DD, hypotonia CA Cerebellar syndrome, stereotyped behaviour, progressive cognitive decline Int. J. Mol. Sci. 2022,23, 11847 9 of 26 Table 2. Cont. Patient Sex Gene Origin Presentation Inheritance Disease (OMIM) Age of Onset Age at Testing Early Symptoms Brain MRI Additional Clinical Features MD-320 Male ITPR1 Senegal de novo AD SCA15 (606658) SCA29 congenital nonprogressive (117360) 3 mo 5 yo DD, hypotonia, gaze-evoked nystagmus CA Cerebellar cortical hyperintensity from 3 yo (upper > lower hemispheres) Ataxia, NPCA, strabismus, ID, mild lower limbs spasticity MD-106 Male ITPR1 Morocco de novo AD <1 yo 6 yo DD, hypotonia CA Cerebellar cortical hyperintensity from 3 yo (upper > lower hemispheres) Ataxia, NPCA, strabismus, gaze-evoked nystagmus, ID, lower limbs spasticity MD-041 Male ITPR1 Spain de novo AD 2 mo 3 yo Gaze-evoked nystagmus, hypotonia CA Cerebellar cortical FLAIR hyperintensity at age of 4 (upper > lower hemispheres) Ataxia, NPCA, strabismus, abnormal ocular movements, ID MD-200 Male KIF1A Spain de novo AD NESCAV syndrome (614255) <1 yo 12 yo DD, microcephaly, nystagmus CA Ataxia, NPCA, lower limbs spasticity, seizures (4 yo), dysphagia, intellectual disability, axonal neuropathy, optic atrophy (9 yo) MD-189 Male KIF1A Portugal de novo AD SPG30 (610357) 12 mo 22 yo DD, visual deficit CA Brain iron deposits from 17 yo Ataxia, NPCA, optic atrophy, stuttering (2 yo), lower limbs spasticity (3 yo), cognitive regression (4 yo), peripheral neuropathy, seizures (14 yo) MD-299 Male KIF1A Spain de novo AD SPG30 (610357) 11 mo 9 yo DD, hypotonia CA Cerebellar cortical and deep WM FLAIR hyperintensity Ataxia (2 yo), NPCA, acquired microcephaly, lower limbs spasticity (3 yo), cognitive impairment, no regression MD-319 Male LRRK2 Morocco Sporadic AR Parkinson disease (607060) 5 yo 14 yo Tremor Normal Hands tremor, cervical tremor, mild bradykinesia, hoarseness voice (13 yo) Int. J. Mol. Sci. 2022,23, 11847 16 of 26 Figure 3. Structural modelling and luciferase assays. ( A , B ) FUCA1 p.R47P. ( A ) Structural modelling on HuFUCA1 subunit. The catalytic and carboxy terminal domains are coloured differently. Localization of R47P is mapped with a red sphere. A molecule of fucose bound to the active site is shown in sticks representation. ( B ) Hexamer of TmFUCA1 (PDB 1ODU). Each subunit is shown with a different colour. The equivalent residue to R47 from HuFUCA1 is indicated by red spheres, to show its participation in the intersubunit surface. ( C – E ) ITPR1 p.Y552C: ( C ) The cryo-EM structure of ITPR1 tetramer (PDB 7LHE) is represented in an orientation along the membrane plane. Each subunit is shown with a different colour. Y552 is mapped in one of the subunits with a red sphere and labelled. ( D , E ) Detail of the IP3 binding site ( D ), PDB 1N4K, and modelling of the Y5552C variant ( E ). The side chain of tyrosine or cytosine at position 552 as well as those from the lysine/arginine cluster coordinating the phosphoryl groups of I3P are shown in stick representation. I3P is also shown in stick representation with carbon atoms coloured in yellow. Oxygen, nitrogen, phosphorus, and sulphur atoms are coloured red, blue, orange, and green respectively. ( F ) Luciferase assays to assess PLEKHG2 p.T53I and PNKD c.-4C>G variants in transfected HEK293T with expression constructs for wild-type (WT) and mutants. The luciferase activity was normalised to the WT. Data represent the mean ± SEM of three independent experiments performed in triplicate. * p< 0.05; n.s., not significant. To investigate PLEKHG2 p.T53I and PNKD c.-4C>G, luciferase analyses were performed. PLEKHG2 is a RhoGTPase that catalyses the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP), activated by heterotrimeric G protein βγ (G βγ ) subunits [ 25 ]. To assess whether the p.T53I mutation affects the activation of the PLEKHG2 downstream signalling pathway, we used a luciferase reporter assay in HEK293 cells under the control of the SRE promoter, whose transcription activity is known to be regulated Int. J. Mol. Sci. 2022,23, 11847 17 of 26 by Rho GTPases. The transcriptional activation capacity expression of the mutated gene was significantly reduced by 25% compared to the control (Figure 3F). PNKD shares a high percentage of sequence identity (43%) with human hydroxyacylglutathione hydrolase (HAGH). The transcriptional activity of PNKD c.-4C>G resulted in a decrease to ~11% compared to the control (Figure 3F). For both variants, we detected altered luciferase activities. These in vitro findings do not guarantee with absolute certainty that the mutated protein has an abnormal activity. Four novel mutations, located on the consensus sequence at splice sites, causing the loss of at least one exon were detected (Figure 4): CASK c.2589+2T>G led to the skipping of exon 26 (84 bp); PDGFB c.602-1G>C to exon 6 plus part of the 3 0 UTR (untranslated region; 153 bp); CPLANE1 c.7588+3A>G to exon 37 (55 bp); and PMPCA c.633+1G>A to exon 6 (101 bp). We performed immunofluorescence assays to determine the subcellular localization of NIPA1 p.P91R (Table 3), using p.G106R as pathogenic control associated with aberrant protein subcellular distribution [ 26 ]. The immunofluorescence assays performed with several cell markers (M6PR, GRP94, and EEA1) revealed partial colocalisation with these markers, although no differences were observed compared to WT (data not shown). Notwithstanding, the expression pattern using Na + , K + ATPase showed that the mutated NIPA1 protein was concentrated in a perinuclear distribution, whereas the WT protein was distributed throughout the cytoplasm (Figure S3), suggesting an abnormal trafficking of mutated proteins as was established for the NIPA1-associated HSP phenotype [26,27]. Int. J. Mol. Sci. 2022,23, 11847 18 of 26 Figure 4. Impact on transcript processing of splicing mutations. Exons are indicated by boxes and introns are represented by horizontal bars (not to scale). Location of specific primers used in RT-PCR is indicated by arrows. Dotted lines represent the anomalous splicing outcome and sequences of the abnormal products are shown (electropherogram). Genomic mutations (in red) are harboured in a heterozygous state and the consensus transcript was also detected in patients’ samples further confirmed by specific band sequencing. ( A – C ) Reverse transcription-polymerase chain reaction (RTPCR) products obtained from RNA of control and patients’ blood samples (agarose electrophoretic results) and schematic representation of the aberrant splicing events. ( A )CASK c.2589+2T>G (MD- 208) induces the complete skipping of exon 26, corresponding to the lower 402 bp band. ( B )PDGFB c.602-1G>C (MD-173) causes the loss of the last exon of the gene plus 28 bp of the contiguous 3 0 untranslated region (3 0 UTR) region presenting a 367 bp aberrant product absent in the healthy control. ( C )CPLANE1 c.7588+3G>A (MD-216) resulted in single exon 37 skipping leading to an alternative transcript 55 bp shorter than the expected. ( D )PMPCA exon 6 minigene assay. Agarose gel shows the band pattern of transcripts obtained after overexpression of pSPL3-derived constructs: wild-type (WT) allele, mutant allele c.633+1G>A and empty vector ø. SD6 and SA2 are the resident exons of the pSPL3 reporting vector. In the absence of an inserted fragment, a product of 263 bp is obtained. The analysed variant causes the skipping of exon 6. bp, base pair; Ct, control; FW, forward primer; RV, reverse primer. 3. Discussion Our clinical series comprised 124 patients with MDs and/or CA. Genes related to NBIA disorders were initially studied in cases with brain iron deposits and/or a complex Int. J. Mol. Sci. 2022,23, 11847 19 of 26 MDs phenotype that included ataxia, spasticity, and/or peripheral neuropathy. This first approach yielded a conclusive diagnosis for 59 patients. Most patients were PKAN (35.96%) or PLAN (12.28%), which represent the two major NBIA types [ 1 ]. Only four patients suffered from other NBIA disorders (mutations in WDR45,FTL or CP). Another PLAN subject (MD-341) who was directly studied by WES-proband, was later diagnosed. Strikingly, no mutations in the remaining NBIA genes were identified in subsequent analyses, which highlights that the abnormal accumulation of brain iron can be due to a primary mutation, but also to the neurodegenerative process that accompanies the disease. The haplotype for the PANK2 locus in Roma patients confirmed that p.T528M was a founder mutation in Spain, just as other mutations have been previously reported for several Mendelian diseases [ 28 – 30 ]. The Roma population makes up around 1.5% of the total Spanish population, thus representing one of the largest Roma communities in the world (http://www.unioromani.org). PANK2 p.T528M likely represents an ancestral mutational event, since Roma PKAN patients from Spain and from Hungary shared the haplotype. The PANK2 p.T528M-associated haplotype was also constructed in the non-Roma Greek heterozygous patients, which may be the outcome of mixing with neighbouring populations. Ultimately, the different haplotypes obtained for the non-Roma Spanish proband heterozygous for PANK2 p.T528M showed that the mutation has arisen in different occasions throughout history. In fact, this has been reported in non-Roma PKAN-affected subjects [31–34]. PANK2 p.T528M seems to be relatively frequent, likely because homozygous individuals present with a milder clinical picture [ 35 ], which may have made its transmission easier through generations in the Roma population. MDs and ataxias are related to hundreds of genes with high phenotypic overlap. We developed a targeted method that tested between 498 genes in its first version [ 14 ] and 517 in its last version, as new genes of interest were reported. Our custom panel MovDisord displayed a capture with uniform coverage and high read depths in all samples, providing a cost-effective alternative, with a diagnosis success rate of 34.54% (19/55 patients). Nowadays, WES is probably the best strategy; WES-trio may yield ~40% of solved cases versus WES-proband with ~28% [ 36 ]. Nonetheless, we achieved a success rate of 70.0% (7/10 patients) and of 33.33% (3/9 patients) using WES-proband or WES-trio, respectively. It is worth mentioning that in the patients investigated by WES-trio, the most common genes were ruled out by the gene panel MovDisord. Altogether, we obtained a success rate of 52.63% (10/19 cases) using WES. In fact, WES-based strategies seem to display a diagnostic ceiling of ~50% [ 37 ], to a certain extent, due to the difficulty of interpreting new variants, hence the relevance of developing additional studies that help to understand their pathological nature. In the sixty-five patients investigated by panel MovDisord and/or WES, seventeen novel variants were identified, one of them a premature stop codon that was assumed to be detrimental. Together with the cases previously reported [ 12 – 17 ], we achieved experimental evidence of damage for 14 of these new genetic changes. Unfortunately, for some mutations, the possible ad hoc analyses were beyond our possibilities. The studies performed for PNKD c.-4C>G and NIPA1 p.P91R deserve a special mention. The luciferase assay revealed that PNKD c.-4C>G, detected in an affected man and in his asymptomatic daughter, may have a decreased transcriptional activity, supporting that the proper activity of the enzyme may be altered. Although we cannot categorically establish the causality of PNKD c.-4C>G, individuals carrying PNKD mutations who remain apparently unaffected have been reported, and in fact, incomplete penetrance seems to be relatively common in paroxysmal nonkinesigenic dyskinesia [ 38 , 39 ]. This phenomenon may explain the absence of clinical signs in the proband’s daughter, although it is also possible that she presents the first symptoms in her forties like her father. NIPA1 p.P91R was analysed by immunofluorescence and we concluded that intracellular trafficking would be impaired, as previously reported for other NIPA1 mutations causing HSP [ 26 , 27 ]. NIPA1 p.P91R was detected in a patient with a complex phenotype, who also suffered from a partial trisomy 18p and a partial monosomy 18q. Known syndromes Int. J. Mol. Sci. 2022,23, 11847 20 of 26 due to a similar chromosomal alteration have nothing to do with MDs [ 40 , 41 ]. A patient with a partial deletion of the chromosome 18q was reported with a complex phenotype, which shared some neurological findings with our proband: intellectual disability, sensorineural hearing loss and dystonia [ 42 ]. It is not possible to establish the contribution of NIPA1 p.P91R to the clinical outcome with the available data. In this cohort, 29 patients with phenotypes of MDs, spasticity, or ataxia were diagnosed with 23 distinct genetic conditions. Of those, five patients presented MDs such as dystonia or parkinsonism, spasticity, and suspicion of brain iron deposits or GP hypointensities; they were associated with variants in FBXO7,FUCA1,GLB1,TPP1, and KIF1A. In fact, isolated cases with GP hypointensities consistent with iron excess are known in at least 17 distinct genes [ 1 ], including AP4M1 and AP4S1, which, like FUCA1,GLB1, and TPP1, are involved in lysosomal disorders. Lysosomes are implicated in the recycling of membranes, autophagy and even iron metabolism. It is tempting to speculate that abnormal iron accumulation may result from a defect in intralysosomal recycling. Patients with NBIA phenotype on MRI and mutations in FUCA1 (1 case) or in GLB1 (4 cases) have been previously described [ 43 – 47 ]. To our knowledge, our proband MD-153 is the first case with the early appearance of GP hypointensities and TPP1 mutations. The three patients here described, MD-137 (FUCA1), MD-020 (GLB1), and MD-153 (TPP1), suffered from lysosomal diseases. Additionally, a subject with α -mannosidosis, also a lysosomal storage disease, who presented hypointensity signals in the GP was published [ 48 ]. Taken together, NBIA on MRI seems not to be so rare in some lysosomal disorders, especially in GM1-gangliosidosis, since so far, five unrelated cases are known (including our proband). Thus, GLB1 has been suggested as an NBIA gene [ 49 ], although this depends on the authors. Single cases with NBIA phenotype have been reported for several genes such as REPS1 or CRAT [ 50 ], and in occasions, they are described as NBIA genes [ 51 ], or as genes to be considered NBIA genes if additional cases are reported [52]. In 22 out of 29 solved patients, the phenotype was associated with CA, but only one was diagnosed with PLAN (MD-341). It is worth mentioning the early appearance of cerebellar cortical hyperintensities in seven patients with a non-progressive phenotype associated to variants in ITPR1 (3), SPTBN2 (2), PMPCA, and KIF1A genes, resembling the neuroimaging pattern recognised in neurodegenerative disorders such as PLAN, mitochondrial disorders, late onset GM2 gangliosidosis, and recently in our patient with PRDX3-associated neurodegeneration (PRAN) [ 17 ]. This paradox has already been described in other patients with ataxia and mutations in KIF1A,ITPR1, and PMPCA but also in KCNC3, and CACNA1A genes [ 53 – 55 ]. It has been presumed that the bright cortex in the cerebellum is a reactive gliosis to the neuronal cell loss and axonal swelling, but different hypotheses are pending to explain how diverse genetic disorders affecting predominantly the cerebellum early in life can present a neurodegenerative sign on neuroimaging, but stable or even improving neurodevelopment over the years. In addition, the three patients with ITPR1 mutations also had a superior CA, a pattern very recently proposed to be related to ITPR1 in 83% of a cohort that included carriers of the p.T267M and p.R269W variants like two of our patients [51]. Globally, our cohort presented a high phenotypic overlap as reported in MDs, ataxias, and NBIA disorders [ 1 , 2 ], with non-classical NBIA genes involved. Interestingly, two probands presented MDs, CA, and brain iron deposits or GP hypointensities, carrying mutations in FBXO7 and TPP1, respectively. Spasticity and CA were a common combination in 10 patients with variants in FBXO7,PLEKHG2,TPP1,ITPR1,KIF1A, and SPG7 genes; ataxia was also present in those with ITPR1,KIF1A, and SPG7 mutations; two probands with mutations in EXOSC3 suffered from early severe encephalopathy and CA, with progressive motor neuron signs after the first years of life. Int. J. Mol. Sci. 2022,23, 11847 21 of 26 4. Material and Methods 4.1. Patients The study was conducted in accordance with the Declaration of Helsinki, and was approved by the Ethic Committees at Hospital Sant Joan de Déu in Barcelona (protocol code PIC-27-15 dated 12 March 2015) and Hospital Universitari i Politècnic La Fe in València (protocol codes: 2019/0052 dated 22 May 2019, and 2021/65/1 dated 22 September 2021). Our cohort comprised 124 paediatric and adult patients with MDs and/or CA. PLAN and PKAN cohorts were previously described [ 5 , 35 ]. Acquired aetiologies were fully excluded in all of them. Demographic and familial history was systematically collected. Phenotyping included standardised assessments of ataxia, MDs, and other neurological symptoms such as spasticity, muscle weakness, or signs of peripheral neuropathy. Based on the phenotypic traits, we classified the patients according to two clinical presentations: (1) MDs such as dystonia, parkinsonism or tremor; spasticity; with or without brain iron deposits; (2) Early-onset ataxia or encephalopathy and CA, with or without brain iron deposits. 4.2. Neuroimaging Approaches Brain MRI studies were performed in 1.5- and 3-T scanners, depending on the referral hospitals. The identification of iron deposit signs in basal ganglia, particularly in globus pallidus (GP) and substantia nigra (SN), was ascertained by iron-sensitive sequences, especially susceptibility weighted imaging (SWI) and T2-weighted imaging (T2WI). In MRI studies without these specific sequences, T2-weighted (T2W) hypointensities in basal ganglia were considered only suspicious with regard to iron deposits, according to normal age-dependent signals [ 3 ]. CA was ascertained by loss of cerebellar volume in at least two consecutive cerebral MRI studies. To assess the progression of CA, a quantitative analysis by mid-sagittal vermis relative diameter (MVRD) was performed in cerebral MRIs [ 5 , 13 ]. Additionally, cerebellar cortical hyperintensities were ascertained on T2-weighted or FLAIR MRI sequences, since they are associated with neurodegenerative phenotypes as PLAN [ 5 ]. 4.3. Genetic and Genomic Studies 4.3.1. Genetic Analysis of NBIA Genes In the first phase, the study was conducted based on the patients’ clinical findings. In 114 patients, the main NBIA genes, PANK2,PLA2G6,WDR45,FTL, and CP, were screened by Sanger sequencing as previously described [ 5 , 35 ], including the analysis of large deletions and/or duplications in PANK2 and PLA2G6 by Multiplex Ligationdependent Probe Amplification (SALSA MLPA probemix P120, MRC Holland, Amsterdam, The Netherlands) (Figure 1). To investigate if the PANK2 c.1583C>T (rs137852967) detected in homozygosis in Roma patients was a founder mutation, we constructed the haplotype for the PANK2 locus. We included eight homozygous Roma probands from Spain (7) and from Hungary (1), and four heterozygous non-Roma probands from Greece (3) and from Spain (1). We selected five extragenic markers (3 microsatellites and 2 SNPs) and three intragenic SNPs from the UCSC Genome Browser (https://genome.ucsc.edu/, accessed on 4 November 2021), the GeneLoc (https://genecards.weizmann.ac.il/geneloc/index.shtml, accessed on 4 November 2021) or the dbSNP (https://www.ncbi.nlm.nih.gov/snp/, accessed on 4 November 2021), which spanned a 1.06 Mb region flanking the PANK2 gene: cen_rs1078152-D20S867-rs6084513-rs137852967-rs6037695-rs7270329-D20S889-D20S116- rs1628323_tel. Protocol has been described previously [ 56 ]. Forward PCR primers (available upon request) were labelled with FAM fluorescent dyes (Sigma Aldrich, St. Louis, MO, USA). Amplicons were electrophoresed using an ABI3730xl (Applied Biosystems, Foster City, CA, USA) and analysed with the Peak Scanner Software v2.0, at the Service of Genomics and Translational Genetics (CIPF, Valencia, Spain). Int. J. Mol. Sci. 2022,23, 11847 22 of 26 4.3.2. Gene Panel, WES and Chromosomal Microarray Analysis The 55 remaining patients without a conclusive result after the analysis of the NBIA genes were investigated using a custom panel MovDisord (Figure 1). The Table S1 shows the 517 genes involved in MDs and ataxia included in the last version of the panel MovDisord, which was reported in its first version [14]. Later, nine remaining patients without genetic diagnoses were investigated by WES- trio, and to compare panel genes versus WES, 10 additional patients were directly investigated by WES-proband as previously reported [ 17 ], except for patient MD-277, which was investigated as previously reported [12]. Bioinformatics analyses (filtering data, study of the novelty of the candidate variants and CNVs, copy number variants) were carried out as previously described [ 17 ]. WES data from unsolved patients were recently reanalysed using the platform RD Connect Genome-Phenome Analysis Platform (https://platform.rd-connect.eu/, accessed on 4 November 2021). Molecular karyotype was performed using genomic DNA from peripheral blood leukocytes andthe CytoScan 750KArray platform (AffymetrixInc., SantaClara, CA, USA) according to the manufacturer’s protocol. Hybridised arrays were scanned on an Affymetrix GeneChip Scanner 3000 and the resulting files were analysed with Chromosome Analysis Suite (ChAS) (Affymetrix Inc., Santa Clara, CA, USA) software v3.1, based on the reference genome sequence of the UCSC Genome Browser hg19. DGV (http://dgv.tcag.ca/dgv/app/home, accessed on 16 July 2021) and DECIPHER (https://decipher.sanger.ac.uk/, accessed on 16 July 2021) were used for the interpretation of CNVs. 4.4. Studies to Investigate the Pathogenicity of Novel Variants 4.4.1. Structural Modelling: ITPR1 and FUCA1 Figure 3was prepared using PYMOL Molecular Graphics System, v2.0 Schrödinger, LLC (https://pymol.org, accessed on 15 September 2021). ITPR1 p.Y552C was modelled in the crystal structure of the IP3 binding domain of ITPR1 (residues 236-602, PDB entry 1N4K) [ 24 ]. FUCA1 p.R47P was modelled by SWISS-MODEL [ 57 ], from the crystal structure of TmFUCA1 (PDB entries 1HL8, 1HL9 and 1ODU) [21]. 4.4.2. Luciferase Reporter Assays: PLEKHG2 and PNKD The reporter vector pGL4.33[luc2P/SRE/Hygro] (Promega, Madison, WI, USA) and PLEKHG2 (NM_022835.3) cloned into the pcDNA3.1:myc (N-ter) vector, kindly ceded by Dr. Chen Songhai [ 25 ], were employed in PLEKHG2 assays. For PNKD, a fragment of 933 bp of the 5’ upstream sequence of the gene was amplified by PCR from a healthy control using the Herculase II Fusion DNA Polymerase (Agilent Technologies, Foster City, CA, USA) and cloned into the pGL4.24[luc2P/minP] reporter vector (Promega, Madison, WI, USA) between XhoI/HindIII restriction sites. The mutants (PLEKHG2 p.T53I and PNKD c.-4C>G) were obtained using the aforementioned technique. Primers are available upon request. HEK293T cells were cultured with complete DMEM (supplemented with 10% heat-inactivated FBS, 5 g/L D-glucose, 1% P/S and 1% L-glutamine) in 24-well plates. The following day, cells were transiently transfected using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA) with the pertinent pGL4 constructs, and renilla luciferase normalisation vector (pRL-TK) with different p.GL4: pRL-TK ratio for each experiment, being 30:1 for PNKD and 3:1 for PLEKHG2. Furthermore, in the case of PLEKHG2, the pGL4.33[luc2P/SRE/Hygro] vector was co-transfected with wild type or mutated PLEKHG2 and after six hours the cell medium was changed to complete DMEM without FBS, as previously described [ 25 , 58 ]. For both assays, after 24 h, luciferase activity was assessed using the Dual-Luciferase ® Reporter Assay System (Promega, Madison, WI, USA). Three independent experiments were carried out for statistical analysis by unpaired two-sample ttest. Int. J. Mol. Sci. 2022,23, 11847 23 of 26 4.4.3. Transcript Analysis and Minigenes: CASK, PDGFB, CPLANE1/C5orf42 and PMPCA Protocols for both approaches are described in Sánchez-Monteagudo et al. [ 59 ]. Primers are available upon request. 4.4.4. NIPA1 Subcellular Location The NIPA1 (NM_144599.4) tagged with FLAG in C-ter and cloned in pcDNA3.1 vector was acquired from GenEZ ™ ORF cDNA Clones (GenScript, Piscataway, NJ, USA). The NIPA1 p.P91R and p.G106R missense mutations were introduced using a Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) using specific primers for each mutation (available upon request). HeLa cells were grown in complete DMEM and transiently transfected with 2 µ g of NIPA1 wild-type (WT), p.P91R, or p.G106R with FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). After 24 h, the cells were fixed, permeabilised and blocked as previously described [ 13 ]. The samples were incubated with the primary antibodies anti-FLAG (Sigma-Aldrich, Saint Louis, MO, USA) and anti-Na + /K + - ATPase, anti-mannose-6-phosphate receptor (M6PR), anti-glucose-regulated protein 94 KDa (GRP94), or anti-early endosome antigen 1 (EEA1; Abcam, Cambridge, UK). The following day, they were exposed to the appropriate secondary antibodies conjugated with fluorophores (Invitrogen, Carlsbad, CA, USA) and examined using the SP2-Leica confocal microscope (Leica, Wetzlar, Germany). 5. Conclusions The NGS technology has made it possible to accelerate the diagnosis of Mendelian MDs, but the number of unsolved cases is still too high. The interpretation of the genetic variants and their phenotypic consequences is a challenge. The low prevalence of each clinical form, the great genetic heterogeneity, and the notable variable expressivity complicate the diagnosis. This report adds new genetic and clinical pieces to the puzzle of MDs and/or ataxias, and contributes to improving the genetic counselling and care of these patients with rare diseases. We have to work hard in order to “enable all people living with a rare disease to receive an accurate diagnosis, care and available therapy within one year of coming to medical attention” [60]. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms231911847/s1. Author Contributions: Funding acquisition, B.P.-D. and C.E.; investigation, D.M.-R., I.H., P.S., N.G.- R., R.B.-F., C.T., C.M.-M., I.M.-C., M.J.M.-G., A.G.-R., R.B.-M., I.S.-B., I.M.-T., A.D.-R., P.J., E.M. (Esther Moreno), L.P.-P., M.O.G., Á.R.-G., E.M. (Esteban Muñoz), M.J.M., A.S.-M., C.F., A.A.-B., R.M.P., S.J.-M., P.M., V.L., B.P.-D., A.D., S.A.-A. and C.E.; writing—original draft, S.A.-A. and C.E.; writing—review and editing, S.A.-A. and C.E. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D+I Plan co-funded with European Regional Development Funds (ERDF) [Grants PI18/00147 and PI21/00103 to CE]; the FundacióLa MaratóTV3 [Grants 20143130 and 20143131 to BPD and CE]; and by the Generalitat Valenciana [Grant PROMETEO/2018/135 to CE]. Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014–2020). PS had an FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport [FPU15/00964]. IH has a PFIS-PhD fellowship [FI19/00072]. ASM has a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data relevant to the study are included in the article or uploaded as supplementary information. Int. J. Mol. Sci. 2022,23, 11847 24 of 26 Acknowledgments: We are in debt with the patients and their families for agreeing to participate in this study. We thank Maria Judit Molnar and Renáta Bencsi who kindly provided a sample of a Hungarian patient carrier of the PANK2 p.T528M. Conflicts of Interest: The authors declare that they have no competing interests. References 1. Hinarejos, I.; Machuca-Arellano, C.; Sancho, P.; Espinos, C. Mitochondrial Dysfunction, Oxidative Stress and Neuroinflammation in Neurodegeneration with Brain Iron Accumulation (NBIA). Antioxidants 2020,9, 1020. [CrossRef] [PubMed] 2. Tello, C.; Darling, A.; Lupo, V.; Pérez-Dueñas, B.; Espinós, C. On the complexity of clinical and molecular bases of neurodegeneration with brain iron accumulation. Clin. Genet. 2018,93, 731–740. [CrossRef] [PubMed] 3. Lee, J.H.; Yun, J.Y.; Gregory, A.; Hogarth, P.; Hayflick, S.J. Brain MRI Pattern Recognition in Neurodegeneration with Brain Iron Accumulation. Front. Neurol. 2020,11, 1024. [CrossRef] 4. Zhou, B.; Westaway, S.K.; Levinson, B.; Johnson, M.A.; Gitschier, J.; Hayflick, S.J. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat. Genet. 2001,28, 345–349. [CrossRef] [PubMed] 5. Darling, A.; Aguilera-Albesa, S.; Tello, C.A.; Serrano, M.; Tomas, M.; Camino-Leon, R.; Fernandez-Ramos, J.; Jimenez-Escrig, A.; Poo, P.; O’Callaghan, M.; et al. PLA2G6-associated neurodegeneration: New insights into brain abnormalities and disease progression. Park. Relat. Disord. 2019,61, 179–186. [CrossRef] 6. Montaut, S.; Tranchant, C.; Drouot, N.; Rudolf, G.; Guissart, C.; Tarabeux, J.; Stemmelen, T.; Velt, A.; Fourrage, C.; Nitschke, P.; et al. Assessment of a Targeted Gene Panel for Identification of Genes Associated with Movement Disorders. JAMA Neurol. 2018,75, 1234–1245. [CrossRef] [PubMed] 7. Sun, M.; Johnson, A.K.; Nelakuditi, V.; Guidugli, L.; Fischer, D.; Arndt, K.; Ma, L.; Sandford, E.; Shakkottai, V.; Boycott, K.; et al. Targeted exome analysis identifies the genetic basis of disease in over 50% of patients with a wide range of ataxia-related phenotypes. Genet. Med. 2018,21, 195–206. [CrossRef] 8. Coutelier, M.; Hammer, M.B.; Stevanin, G.; Monin, M.L.; Davoine, C.S.; Mochel, F.; Labauge, P.; Ewenczyk, C.; Ding, J.; Gibbs, J.R.; et al. Efficacy of Exome-Targeted Capture Sequencing to Detect Mutations in Known Cerebellar Ataxia Genes. JAMA Neurol. 2018,75, 591–599. [CrossRef] 9. Elert-Dobkowska, E.; Stepniak, I.; Krysa, W.; Ziora-Jakutowicz, K.; Rakowicz, M.; Sobanska, A.; Pilch, J.; Antczak-Marach, D.; Zaremba, J.; Sulek, A. Next-generation sequencing study reveals the broader variant spectrum of hereditary spastic paraplegia and related phenotypes. Neurogenetics 2019,20, 27–38. [CrossRef] 10. D’Amore, A.; Tessa, A.; Casali, C.; Dotti, M.T.; Filla, A.; Silvestri, G.; Antenora, A.; Astrea, G.; Barghigiani, M.; Battini, R.; et al. Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study. Front. Neurol. 2018 ,9, 981. [CrossRef] [PubMed] 11. Richards, S.; Aziz, N.; Bale, S.; Bick, D.; Das, S.; Gastier-Foster, J.; Grody, W.W.; Hegde, M.; Lyon, E.; Spector, E.; et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015,17, 405–424. [CrossRef] [PubMed] 12. Jesús, S.; Hinarejos, I.; Carrillo, F.; Martínez-Rubio, M.D.; Macías-García, D.; Sánchez-Monteagudo, A.; Adarmes, A.; Lupo, V.; Pérez-Dueñas, B.; Mir, P.; et al. NR4A2 is involved in dystonia-parkinsonism with intellectual disability, language impairment and motor tics. Neurol. Genet. 2021,7, e543. [CrossRef] 13. Sancho, P.; Andrés-Bordería, A.; Gorría-Redondo, N.; Llano, K.; Martínez-Rubio, D.; Yoldi-Petri, M.E.; Blumkin, L.; Rodríguez de la Fuente, P.; Gil-Ortiz, F.; Fernández-Murga, L.; et al. Expanding the β -III spectrin-associated phenotypes toward non-progressive congenital ataxias with neurodegeneration. Int. J. Mol. Sci. 2021,22, 2505. [CrossRef] [PubMed] 14. Correa-Vela, M.; Lupo, V.; Montpeyo, M.; Sancho, P.; Marce-Grau, A.; Hernandez-Vara, J.; Darling, A.; Jenkins, A.; Fernandez- Rodriguez, S.; Tello, C.; et al. Impaired proteasome activity and neurodegeneration with brain iron accumulation in FBXO7 defect. Ann. Clin. Transl. Neurol. 2020,7, 1436–1442. [CrossRef] 15. Campins-Romeu, M.; Baviera-Munoz, R.; Sastre-Bataller, I.; Bataller, L.; Jaijo, T.; Martinez-Torres, I. Hereditary Spastic Paraplegia 7 Presenting as Multifocal Dystonia with Prominent Cranio-Cervical Involvement. Mov. Disord. Clin. Pract. 2021 ,8, 966–968. [CrossRef] 16. Baviera-Munoz, R.; Martinez-Rubio, D.; Sastre-Bataller, I.; Campins-Romeu, M.; Losada-Lopez, M.; Perez-Garcia, J.; Novella- Maestre, E.; Martinez-Torres, I.; Espinos, C. A 3.9-Mb Deletion on 2p11.2 Comprising the REEP1 Gene Causes Early-Onset Atypical Parkinsonism. Neurol. Genet. 2021,7, e642. [CrossRef] [PubMed] 17. Martinez-Rubio, D.; Rodriguez-Prieto, A.; Sancho, P.; Navarro-Gonzalez, C.; Gorria-Redondo, N.; Miquel-Leal, J.; Marco-Marin, C.; Jenkins, A.; Soriano-Navarro, M.; Hernandez, A.; et al. Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3. Hum. Mol. Genet. 2022, ddac146. [CrossRef] [PubMed] 18. Bertini, E.; Zanni, G.; Boltshauser, E. Nonprogressive congenital ataxias. Handb Clin. Neurol. 2018,155, 91–103. [PubMed] 19. Baviera-Munoz, R.; Campins-Romeu, M.; Carretero-Vilarroig, L.; Sastre-Bataller, I.; Martinez-Torres, I.; Vazquez-Costa, J.F.; Muelas, N.; Sevilla, T.; Vilchez, J.J.; Aller, E.; et al. Clinical and genetic characteristics of 21 Spanish patients with biallelic pathogenic SPG7 mutations. J. Neurol. Sci. 2021,429, 118062. [CrossRef] [PubMed] Int. J. Mol. Sci. 2022,23, 11847 25 of 26 20. Jonch, A.E.; Douard, E.; Moreau, C.; Van Dijck, A.; Passeggeri, M.; Kooy, F.; Puechberty, J.; Campbell, C.; Sanlaville, D.; Lefroy, H.; et al. Estimating the effect size of the 15q11.2 BP1-BP2 deletion and its contribution to neurodevelopmental symptoms: Recommendations for practice. J. Med. Genet. 2019,56, 701–710. [CrossRef] 21. Sulzenbacher, G.; Bignon, C.; Nishimura, T.; Tarling, C.A.; Withers, S.G.; Henrissat, B.; Bourne, Y. Crystal structure of Thermotoga maritima alpha-L-fucosidase. Insights into the catalytic mechanism and the molecular basis for fucosidosis. J. Biol. Chem. 2004,279, 13119–13128. [CrossRef] [PubMed] 22. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Zidek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021,596, 583–589. [CrossRef] [PubMed] 23. Fan, G.; Baker, M.L.; Wang, Z.; Baker, M.R.; Sinyagovskiy, P.A.; Chiu, W.; Ludtke, S.J.; Serysheva, I.I. Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature 2015,527, 336–341. [CrossRef] [PubMed] 24. Bosanac, I.; Alattia, J.R.; Mal, T.K.; Chan, J.; Talarico, S.; Tong, F.K.; Tong, K.I.; Yoshikawa, F.; Furuichi, T.; Iwai, M.; et al. Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 2002 ,420, 696–700. [CrossRef] [PubMed] 25. Edvardson, S.; Wang, H.; Dor, T.; Atawneh, O.; Yaacov, B.; Gartner, J.; Cinnamon, Y.; Chen, S.; Elpeleg, O. Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization. Neurogenetics 2016,17, 25–30. [CrossRef] 26. Zhao, J.; Matthies, D.S.; Botzolakis, E.J.; Macdonald, R.L.; Blakely, R.D.; Hedera, P. Hereditary spastic paraplegia-associated mutations in the NIPA1 gene and its Caenorhabditis elegans homolog trigger neural degeneration in vitro and in vivo through a gain-of-function mechanism. J. Neurosci. 2008,28, 13938–13951. [CrossRef] 27. Goytain, A.; Hines, R.M.; El-Husseini, A.; Quamme, G.A. NIPA1(SPG6), the basis for autosomal dominant form of hereditary spastic paraplegia, encodes a functional Mg2+ transporter. J. Biol. Chem. 2007,282, 8060–8088. [CrossRef] 28. Sevilla, T.; Martinez-Rubio, D.; Marquez, C.; Paradas, C.; Colomer, J.; Jaijo, T.; Millan, J.M.; Palau, F.; Espinos, C. Genetics of the Charcot-Marie-Tooth disease in the Spanish Gypsy population: The hereditary motor and sensory neuropathy-Russe in depth. Clin. Genet. 2013,83, 565–570. [CrossRef] 29. Casana, P.; Martinez, F.; Haya, S.; Lorenzo, J.I.; Espinos, C.; Aznar, J.A. Q1311X: A novel nonsense mutation of putative ancient origin in the von Willebrand factor gene. Br. J. Haematol. 2000,111, 552–555. 30. Gil-Pena, H.; Coto, E.; Santos, F.; Espino, M.; Cea Crespo, J.M.; Chantzopoulos, G.; Komianou, F.; Gomez, J.; Alonso, B.; Iglesias, S.; et al. A new SLC12A3 founder mutation (p.Val647Met) in Gitelman’s syndrome patients of Roma ancestry. Nefrologia 2017,37, 423–428. [CrossRef] 31. Wu, Y.W.; Hess, C.P.; Singhal, N.S.; Groden, C.; Toro, C. Idiopathic basal ganglia calcifications: An atypical presentation of PKAN. Pediatr. Neurol. 2013,49, 351–354. [CrossRef] 32. Dusek, P.; Tovar Martinez, E.M.; Madai, V.I.; Jech, R.; Sobesky, J.; Paul, F.; Niendorf, T.; Wuerfel, J.; Schneider, S.A. 7-Tesla Magnetic Resonance Imaging for Brain Iron Quantification in Homozygous and Heterozygous PANK2 Mutation Carriers. Mov. Disord. Clin. Pract. 2014,1, 329–335. [CrossRef] [PubMed] 33. Akcakaya, N.H.; Iseri, S.U.; Bilir, B.; Battaloglu, E.; Tekturk, P.; Gultekin, M.; Akar, G.; Yigiter, R.; Hanagasi, H.; Alp, R.; et al. Clinical and genetic features of PKAN patients in a tertiary centre in Turkey. Clin. Neurol. Neurosurg. 2017 ,154, 34–42. [CrossRef] [PubMed] 34. Yapici, Z.; Akcakaya, N.H.; Tekturk, P.; Iseri, S.A.; Ozbek, U. A novel gene mutation in PANK2 in a patient with severe jaw-opening dystonia. Brain Dev. 2016,38, 755–758. [CrossRef] 35. Darling, A.; Tello, C.; Martí, M.J.; Garrido, C.; Aguilera-Albesa, S.; Tomás Vila, M.; Gastón, I.; Madruga, M.; González Gutiérrez, L.; Ramos Lizana, J.; et al. Clinical rating scale for pantothenate kinase-associated neurodegeneration: A pilot study. Mov. Disord. 2017,32, 1620–1630. [CrossRef] [PubMed] 36. Wright, C.F.; FitzPatrick, D.R.; Firth, H.V. Paediatric genomics: Diagnosing rare disease in children. Nat. Rev. Genet. 2018 ,19, 325. [CrossRef] 37. Ngo, K.J.; Rexach, J.E.; Lee, H.; Petty, L.E.; Perlman, S.; Valera, J.M.; Deignan, J.L.; Mao, Y.; Aker, M.; Posey, J.E.; et al. A diagnostic ceiling for exome sequencing in cerebellar ataxia and related neurological disorders. Hum. Mutat. 2020 ,41, 487–501. [CrossRef] [PubMed] 38. Djarmati, A.; Svetel, M.; Momcilovic, D.; Kostic, V.; Klein, C. Significance of recurrent mutations in the myofibrillogenesis regulator 1 gene. Arch. Neurol. 2005,62, 1641. [CrossRef] [PubMed] 39. Panedey, S.; Ramkumarsingh, L.; Mahadevan, L. Progressive nonparoxysmal chorea and dystonia due to myofibrillogenesis regulator-1 gene mutation. Park. Relat. Disord. 2019,60, 186–187. [CrossRef] 40. Bartels, E.; Draaken, B.; Kazmierczak, B.; Spranger, S.; Schramm, C.; Baudisch, F.; Nöthen, M.M.; Schmiedeke, E.; Ludwig, M.; Reutter, H. De novo partial trisomy 18p and partial monosomy 18q in a patient with anorectal malformation. Cytogenet. Genome Res. 2011,134, 243–248. 41. Hu, H.; Hao, J.; Yao, H.; Chang, Q.; Li, R.; Zhang, X.; Liang, Z. Prenatal diagnosis of de novo partial trisomy 18p and partial monosomy 18q recurrent in a family with fatal aortic coarctation. Gene 2013,517, 132–136. [CrossRef] [PubMed] 42. Gordon, M.F.; Bressman, S.; Brin, M.F.; de Leon, D.; Warburton, D.; Yeboa, K.; Fahn, S. Dystonia in a patient with deletion of 18q. Mov. Disord. 1995,10, 496–499. [CrossRef] 43. Gautschi, M.; Merlini, L.; Calza, A.M.; Hayflick, S.; Nuoffer, J.M.; Fluss, J. Late diagnosis of fucosidosis in a child with progressive fixed dystonia, bilateral pallidal lesions and red spots on the skin. Eur. J. Paediatr. Neurol. 2014 ,18, 516–519. [CrossRef] [PubMed]