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See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CASE STUDY Novel PLEKHG5 mutations in a patient with childhood-onset lower motor neuron disease Lidia Gonzalez-Quereda1,2 , Inmaculada Pagola3, Pablo Fuentes Prior4, Sara Bernal1,2, Maria Jose 3, Josefa Salgado Garrido5, Pia Gallano1,2 & Ivonne Jerico Rodriguez1, Laura Torne 3 1 Genetics Department, IIB Sant Pau, Hospital de Sant Pau, Barcelona, 08041, Spain 2 U705 CIBERER, Instituto de Salud Carlos III, Madrid, 28029, Spain 3 Neurology Department, Complejo Universitario de Navarra, IdisNa, Navarra, 31008, Spain 4 Molecular Bases of Disease, Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, 08041, Spain 5 Genomic Medicine, Navarrabiomed, Complejo Hospitalario de Navarra (CHN)-Universidad Pu blica de Navarra (UPNA), IdisNa, Pamplona, 31008, Spain Correspondence Abstract Lidia Gonzalez-Quereda, Genetics Department, IIB Sant Pau, Sant Quintı, St., 77 The PLEKHG5 gene encodes a protein that activates the nuclear factor kappa B P2-010, Barcelona 08041, Spain. Tel: +34 (NFjB) signaling pathway. Mutations in this gene have been associated with 932919000 Ext. 8073; E-mail: distal spinal muscular atrophy IV and intermediate axonal neuropathy C, both lgonzalezq@santpau.cat with an autosomal recessive mode of inheritance. Two families with low motor neuron disease (LMND) caused by mutations in PLEKHG5 have been reported to date. We present a third LMND family, the first nonconsanguineous, due to Funding Information This study was granted by FIS PI18/01585, two not previously reported PLEKHG5 mutations. Our results confirm and funded by ISCIII and FEDER, “Una manera de extend previous findings linking PLEKHG5 mutations to lower motor neuron hacer Europa” and by Fundacio n Mutua diseases. Madrilen~a in the “Convocatoria de Ayudas a n en Salud 2015”. la Investigacio Received: 22 September 2020; Revised: 9 November 2020; Accepted: 11 November 2020 Annals of Clinical and Translational Neurology 2021; 8(1): 294–299 doi: 10.1002/acn3.51265 611067) and intermediate axonal neuropathy C Background (CMTRIC; MIM 615376), both of which have an autoso- PLEKHG5 (pleckstrin homology and RhoGEF domain mal recessive pattern of inheritance.4,6–10 containing G5), codes for a guanine exchange factor Inherited lower motor neuron diseases (LMNDs) (GEF) specifically expressed in motor neurons, where it encompass a genetically heterogeneous group of clinical regulates autophagy of synaptic vesicles. Furthermore, disorders affecting the distal motor nerve up to the level PLEKHG5 plays a role in the activation and regulation of of the anterior horn cell. They are characterized by pro- RhoA signaling pathways, thereby controlling neuronal gressive muscle weakness (distal or proximal) with atro- cell differentiation and maintaining stable cell-cell con- phy and hypo/areflexia, without sensory involvement. tacts. In addition, PLEKHG5 is required for the polarity Classification of these syndromes is complex and not of actively migrating brain cells, but also breast tumor standardized, but a variety of hereditary causes is recog- cells.1–3 Mutations in PLEKHG5 appear to be involved in nized. Differential diagnosis between LMNDs is difficult cell death through impaired NF-jB signaling,4,5 and are because symptoms often overlap and phenotypes and responsible for different forms of motor neuron diseases genotypes vary widely. With the development of next- such as distal spinal muscular atrophy IV (DSMA4; MIM generation sequencing (NGS) the number of genetic 294 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 23289503, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51265 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License L. Gonzalez-Quereda et al. LMND Case Related to PLEKHG5 Mutations causes of LMNDs is expanding, and novel genetic variants Lab tests revealed mild hyperCKemia (two to threefold are being identified. Several causative genes have been above the normal range). Muscle Computerized Tomog- reported, with varying clinical manifestations, mode of raphy (CT) showed a predominant involvement of glutei inheritance, and age of onset.11–13 and anterior muscle group at thighs (Figure 1). To date, LMND caused by mutations in PLEKHG5 has Nerve conduction studies were normal and the needle been reported in two families.4,8,9 Here we present the EMG showed denervation with the presence of fibrillation first nonconsanguineous LMND case due to two potentials, motor unit action potentials (MUAPs) with PLEKHG5 frameshift mutations not previously described. increased duration and amplitude, and a reduced recruit- ment pattern. The muscle biopsy was consistent with a denervation pattern. A clinical diagnosis of lower motor Case Presentation neuron disease was established. A 33-year-old Spanish male presented with a history of pro- gressive and symmetric proximal and distal limb weakness, Methods predominantly in the legs, since age 8, and loss of ambula- tion at age 17. He was an only child and the family had no DNA was extracted from peripheral blood following stan- history of consanguinity. Physical examination showed sca- dard procedures. The SMN1 gene was analyzed by MPLA pula winging and the absence of deep reflexes. Facial (SMA Salsa Kit P021, MRC Holland, Netherlands) and involvement and upper motor neuron signs were absent. sequencing of the whole-coding region. We sequenced the Hyperlordosis and contractures of hips and elbows were also proband’s DNA using a custom-targeted NGS gene panel noted. No respiratory or bulbar impairment was observed. including 116 neuromuscular disease-related genes, Figure 1. Axial computed tomography (CT) images of the pelvis (A), thigh (B), and lower leg (C) of the studied patient. Note the different degrees of fatty degeneration (with low density). The most affected muscles are the glutei (thin white arrow in A), and anterior muscle group (thick white arrow in B). In contrast, muscles of the lower legs are comparatively preserved (C). ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 295 23289503, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51265 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LMND Case Related to PLEKHG5 Mutations L. Gonzalez-Quereda et al. Figure 2. Schematic representation of the domain organization in PLEKHG5 (A), showing the position of mutations identified in our patient (boxed) and in previous reports. Mutations described in LMND and iCMT patients are indicated in red and purple, respectively. RDB: Rnd3-binding domain; DH: Dbl homology domain; PH: Pleckstrin homology domain; PDZ: PDZ binding motif; YXXΦ: tyrosine-based sorting motif; Nt: N- terminus; Ct: Ct-terminus. The red box indicates an extremely acidic Glu-rich stretch in the wild-type protein, which is replaced by a highly basic sequence in the p.Leu686Argfs*79 mutant. Green boxes highlight putative SH3-binding motifs. Ser/Thr phosphorylation sites are marked with triangles. Sequence electropherograms showing the patient mutations: c.2057delT in heterozygosis in exon 19 of PLEKHG5 (B); c.2752_2753delGG in heterozygosis in exon 20 of PLEKHG5 (C). Model of the PLEKHG5-RhoA complex showing the reported mutants in the PH domain (D). We used a numbering system based on PLEKHG5 isoform a. For the numbering system favored in protein databases, add +56. following Nextera Rapid Capture technology (Illumina, Ethics Committee at Hospital de la Santa Creu i Sant Pau San Diego, CA). We used 50 ng of genomic DNA to con- (HSCSP) and was performed in accordance with the ethi- struct the library following the manufacturer’s protocols. cal principles of the Helsinki Declaration (WMA, 2013). Sequencing was performed using the MiSeq Platform (Illumina). Variant annotation and filtering were under- Results taken using Illumina Variant Studio 3.0 (Illumina), start- ing with variant call format (VCF) files from each sample. SMN1 gene analysis did not reveal any disease-causative Selected variants had a read depth above 309 and a vari- mutation. However, the custom-targeted NGS gene panel ant frequency reported in Thousand Genomes, Exome identified two novel compound heterozygous frameshift Aggregation Consortium (ExAC), Genome Aggregation mutations in PLEKHG5 as the best candidate variants Database (gnomAD), and Exome Variant Server (EVS) responsible for the clinical findings: 1) c.2057del below 5%. Variant pathogenicity was assessed according (p.Leu686Argfs*79) located in exon 19; and 2) to the American College of Medical Genetics (ACMG) c.2752_2753del (p. Gly918Leufs*16), in exon 20 (Fig- guidelines.14 Clinically relevant variants were confirmed ure 2). This finding was corroborated by the patient’s by Sanger sequencing and segregated in the family. participation in the Navarra 1000 Genomes project PLEKHG5 mutations were named based on RefSeq (NAGEN 1000). Segregation analysis concluded that NM_020631.4 in agreement with previous reports. c.2057del was inherited from the paternal allele, whereas Written informed consent was obtained from the patient c.2752_2753del was inherited from the maternal allele, and his parents. The genetic study was approved by the confirming their recessive inheritance. 296 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 23289503, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51265 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License L. Gonzalez-Quereda et al. LMND Case Related to PLEKHG5 Mutations Table 1. Summary of the reported PLEKHG5 reported cases PLEKHG5 Phenotype/ Age Family mutations* at onset Clinical findingsa Structural and functional impact 1 p.Phe647Ser Severe LMND/ 2 Proximal and distal muscle weakness Unfolding of PH domain, likely followed by p.Phe647Ser to 3.5 years predominating at the lower limbs with protein degradation. generalized areflexia. Contractures, severe scoliosis, and hyperlordosis. Loss of ambulation at the mean age of 8.5 years. Assisted ventilation is needed at a mean age of 16.5 years. Muscle denervation and slightly decreased motor nerve conduction velocities. Mild LMND/ Moderate generalized muscle weakness and 11.5 years slow course of the disease. Hyperlordosis and bilateral winging of scapula. All deep tendon reflexes were abolished. Muscle denervation and subnormal motor and sensory nerve conduction velocities. 2 p.Thr663Met CMTRIC/ 8 years Muscle weakness and atrophy of bilateral distal No major structural impact. Might disrupt p.Gly820Arg muscles, predominantly at lower limbs. binding site for putative macromolecular Bilateral pes cavus and stepage gait. partner. Decreased sensitivity. Decreased motor and Loss of binding sites for regulatory factors sensory nerve conduction velocities. (e.g., PDZ and SH3-containing proteins). 3 p.Pro13Hisfs*45 CMTRIC/ 7 years Limb-girdle muscle weakness and atrophy, foot No functional protein was produced. p.Pro13Hisfs*45 and 20 years deformities, moderately decreased nerve conduction velocities. 4 p.Glu307* CMTRIC/ Limb-girdle muscle weakness and atrophy, foot Truncated protein produced that lacks major p.Glu307* Between the deformities, moderately decreased nerve functional domains (DH/ PH); likely degraded. second and the conduction velocities, loss of large myelinated fifth decade. fibers, and thin myelination 5 p.Pro630His LMND/ 13 years Lower motor neuron type of disease with slow Unfolding of PH domain, likely followed by p.Pro630His progression protein degradation. 6 p.Leu686Argfs*79 LMND/ 8 years Progressive and symmetric proximal and distal Loss of binding sites for regulatory factors p.Gly918Leufs*16 limb weakness, predominantly in the legs. (e.g., PDZ and SH3-containing proteins). In Loss of ambulation at age 17. Scapula p.Leu686Argfs*79, additional loss of acidic winging and absence of deep reflexes. stretch and of several phosphorylation sites. Hyperlordosis and contractures of hips and elbows. Mild hiperCKemia. Denervation pattern with normal nerve conduction studies. a Clinical and molecular findings have been compiled from the following references: Family 1: Maystadt I, et al. Neurology 2006 and Maystadt I et al. AJHG 2007. Family 2: Kim HJ, et al. Orphanet J. Rare Dis. 2013. Family 3 and 4: Azzedine H, et al. Hum. Mol. Genet. 2013. Family 5: € g Ozo uz A, et al. Neurobiol. Aging 2015. Family 6: the present work. chain of Leu686 might engage in Van-der-Waals inter- Discussion actions with other residues of this domain (Figure 2D), Six different PLEKHG5 mutations have been described in its nonconservative exchange by an arginine is unlikely five different families to date, two of which presented to have a significant structural impact. Replacement of LMND,4,9 whereas the other three presented intermediate the C-terminal region by a fully unrelated peptide Charcot-Marie-Tooth (iCMT) forms7,10 (Figure 2A and seems to have much more important functional conse- Table 1). Here we describe a third family with childhood- quences. Most notably, there is a reversal-of-charge in onset and autosomal recessive inheritance of LMND, the downstream Glu705-Asp726 stretch of the protein which is apparently caused by two novel compound (Figure 2A), from extremely acidic (mostly glutamate heterozygous frameshift mutations in PLEKHG5. residues) to highly basic (mostly arginines). This polar- Mutation p.Leu686Argfs*79 affects a residue that ity change is likely to result in impaired interactions of marks the C-terminal end of the PH domain and the PLEKHG5 with phospholipid membranes. This is in start of a long region predicted to be intrinsically dis- addition to the loss of several additional protein–pro- ordered in solution. Even though the aliphatic side tein binding sites such as the tyrosine-based sorting ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 297 23289503, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51265 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LMND Case Related to PLEKHG5 Mutations L. Gonzalez-Quereda et al. motif, Tyr819-Xxx-Xxx-Leu822, responsible for the Further studies are needed to better understand the interaction with the µ-subunit of Adaptor Protein com- pathogenic mechanisms of PLEKHG5 and the different plex as well as Ser/Thr phosphosites at positions 729, clinical manifestations of its mutations (e.g., LMND in 734, 851, 876, and 881. some patients and iCMT in others). Interestingly, other Mutation p.Gly918Leufs*16 replaces the last C-terminal PH domain-containing proteins have also been linked to 89 residues of the protein by an unrelated, shorter 16- neurodegenerative diseases in humans. For instance, residue peptide. Both mutants lack the C-terminal PDZ- mutations in the C-terminal half of dynamin 2 (DNM2) binding motif, Leu1001-Val1006, conserved in all iso- PH domain are associated with a dominant myopathic forms of the protein but isoform 4. These truncated phenotype of centronuclear myopathy [MIM 160150], PLEKHG5 variants also lack several proline-rich stretches whereas mutations in the N-terminal half of the same that are binding motifs for SH3-containing proteins, most domain cause dominant type B iCMT.16 notably the Arg967-Pro973 heptapeptide, which could The high-sequencing output of NGS techniques has interact with both class I and class III SH3 domains. Alto- allowed the identification of new disease-causing muta- gether, the C-terminal region of this GEF appears to be tions thus furthering our understanding of the genetic ba- involved in critically important interactions with other sis of LMNDs. However, many LMND patients do not macromolecules, including perhaps specific membrane yet have a conclusive genetic diagnosis. The detailed clini- domains, and impaired binding to these interactors would cal and molecular analysis of these cases can contribute to explain the observed phenotype. PLEKGH5 regulates establish genotype–phenotype correlations, adding to our autophagy of synaptic vesicles in the axon terminal of understanding of the pathogenic pathways underlying this motor neurons, leading to an accumulation of synaptic heterogenous group of diseases. proteins and resulting in dysfunction of neuromuscular junctions and motoneurons.15 Conceivable, the two muta- Acknowledgments tions identified in the current work impair protein–pro- tein and/or protein-membrane interactions needed during We thank the patient and their relatives for their partici- the complex autophagy process. Since these data come pation in this study. We also thank Carolyn Newey for from in silico predictions, it would be desirable to per- proofreading the text. form functional tests to analyze the stability of the protein and the functional impact of the identified mutations. Authors’ Contributions Unfortunately, we do not have a tissue sample from the patient to perform these analyses. LGQ contributed to the interpretation of the findings, By contrast, the causative mutations in other LMND wrote, and revised the draft of the manuscript. PFP per- families previously reported, p.Pro630His and p.Phe647- formed the structural analyses, revised, and edited the Ser, do affect the core of the PH domain and would pro- manuscript. IP was involved in the acquisition and inter- foundly impair its folding4,9 (Figure 2D). Also along these pretation of imaging data and drafting and revising the lines, missense mutations in the PH domain of PLEKHG5 manuscript. MJR and JSG performed the genetic analysis. (p.Thr663Met) and in the C-terminal region SB contributed to the analysis and interpretation of the (p.Gly820Arg) have also been linked to autosomal-reces- genetic findings. LT performed a bibliographic search. PG sive iCMT disease.7 Thr663 is relatively exposed to bulk contributed to the interpretation of the findings and solvent, and the mutation is unlikely to cause major revised and edited the manuscript. IJ examined the structural rearrangements in the PH domain. However, patient and collected all the clinical data, wrote, and its proximity to Phe647 might point to the disruption of revised the draft of the manuscript. a binding site for a PLEKHG5 cofactor, perhaps Rab26 itself (Figure 2D). On the other hand, mutant Conflict of interest p.Gly820Arg maps to the Tyr-based sorting motif, Y819- L822. However, Arg820 might actually be preferred at the All authors declare that they have no conflict of interest. second position of this motif, as the basic residue is repeatedly found in internalized proteins such as the EGF References receptor. These patients expressed low levels of PLEKHG5 1. Marx R, Henderson J, Wang J, Baraban JM. Tech: A RhoA in the distal sural nerve and in vitro studies suggested GEF selectively expressed in hippocampal and cortical defects in the activation of the NF-kB signaling pathway neurons. J Neurochem 2005;92(4):850–858. as likely explanation for the deleterious effect of the iden- 2. Ngok SP, Geyer R, Kourtidis A, et al. Phosphorylation- tified PLEKHG5 mutants. mediated 14-3-3 protein binding regulates the function of 298 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 23289503, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51265 by Readcube (Labtiva Inc.), Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License L. Gonzalez-Quereda et al. LMND Case Related to PLEKHG5 Mutations the rho-specific guanine nucleotide exchange factor 10. Azzedine H, Zavadakova P, Plante-Bordeneuve V, et al. (RhoGEF) syx. J Biol Chem 2013;288(9):6640–6650. pLEKHG5 deficiency leads to an intermediate form of 3. Dachsel JC, Ngok SP, Lewis-Tuffin LJ, et al. The rho autosomal-recessive charcot-marie-tooth disease. Hum guanine nucleotide exchange factor Syx regulates the Mol Genet 2013;22(20):4224–4232. balance of dia and ROCK activities to promote polarized- 11. Peeters K, Chamova T, Jordanova A. Clinical and genetic cancer-cell migration. Mol Cell Biol 2013;33(24):4909– diversity of SMN1-negative proximal spinal muscular 4918. atrophies. Brain 2014;137(Pt 11):2879–2896. 4. Maystadt I, Rezs€ohazy R, Barkats M, et al. The nuclear 12. Irobi J, De Jonghe P, Timmerman V. Molecular genetics factor jB-activator gene PLEKHG5 is mutated in a form of distal hereditary motor neuropathies. Hum Mol Genet of autosomal recessive lower motor neuron disease with 2004;13(Spec No 2):R195–R202. childhood onset. Am J Hum Genet 2007;81(1):67–76. 13. Zerres K, Rudnik-Sch€ oneborn S. 93rd ENMC international 5. De Toledo M, Coulon V, Schmidt S, et al. The gene for a workshop: Non-5q-spinal muscular atrophies (SMA) - new brain specific RhoA exchange factor maps to the Clinical picture (6–8 April 2001, Naarden, The highly unstable chromosomal region 1p36.2-1p36.3. Netherlands). In: Neuromuscular Disorders 2nd ed. vol. Oncogene 2001;20(50):7307–7317. 13, pp. 179–183. 2003. 6. Liu L, Zhang R. Intermediate charcot-marie-tooth disease. 14. Richards S, Aziz N, Bale S, et al. Standards and guidelines Neurosci Bull 2014;30(6):999–1009. for the interpretation of sequence variants: A joint 7. Kim HJ, Bin HY, Park JM, et al. Mutations in the consensus recommendation of the American College of PLEKHG5 gene is relevant with autosomal recessive Medical Genetics and Genomics and the Association for intermediate Charcot-Marie-Tooth disease Orphanet J Molecular Pathology. Genet Med 2015;17(5):405–424. Rare Dis 2013;8(104). 15. L€uningschr€or P, Binotti B, Dombert B, et al. Plekhg5- 8. Maystadt I, Zarhrate M, Leclair-Richard D, et al. A gene regulated autophagy of synaptic vesicles reveals a for an autosomal recessive lower motor neuron disease pathogenic mechanism in motoneuron disease. Nat with childhood onset maps to 1p36. Neurology 2006;67 Commun 2017;8(1):678. (1):120–124. 16. Kenniston JA, Lemmon MA. Dynamin GTPase regulation 9. € guz A, Uyan O, Ozo € Birdal G, et al. The distinct genetic is altered by PH domain mutations found in pattern of ALS in Turkey and novel mutations. Neurobiol centronuclear myopathy patients. EMBO J 2010;29 Aging 2015;36(4):1764.e9–1764.e18. (18):3054–3067. ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 299