García-Hernández et al. Human Genomics (2021) 15:11 https://doi.org/10.1186/s40246-021-00309-4 PRIMARY RESEARCH Open Access Pathogenic convergence of CNVs in genes functionally associated to a severe neuromotor developmental delay syndrome Juan L. García-Hernández1,2 , Luis A. Corchete2,3 , Íñigo Marcos-Alcalde4,5 , Paulino Gómez-Puertas4 , Carmen Fons6* and Pedro A. Lazo1,2* Abstract Background: Complex developmental encephalopathy syndromes might be the consequence of unknown genetic alterations that are likely to contribute to the full neurological phenotype as a consequence of pathogenic gene combinations. Methods: To identify the additional genetic contribution to the neurological phenotype, we studied as a test case a boy, with a KCNQ2 exon-7 partial duplication, by single-nucleotide polymorphism (SNP) microarray to detect copy-number variations (CNVs). Results: The proband presented a cerebral palsy like syndrome with a severe motor and developmental encephalopathy. The SNP array analysis detected in the proband several de novo CNVs, nine partial gene losses (LRRC55, PCDH9, NALCN, RYR3, ELAVL2, CDH13, ATP1A2, SLC17A5, ANO3), and two partial gene duplications (PCDH19, EFNA5). The biological functions of these genes are associated with ion channels such as calcium, chloride, sodium, and potassium with several membrane proteins implicated in neural cell-cell interactions, synaptic transmission, and axon guidance. Pathogenically, these functions can be associated to cerebral palsy, seizures, dystonia, epileptic crisis, and motor neuron dysfunction, all present in the patient. Conclusions: Severe motor and developmental encephalopathy syndromes of unknown origin can be the result of a phenotypic convergence by combination of several genetic alterations in genes whose physiological function contributes to the neurological pathogenic mechanism. Keywords: CNV, Variome, Dystonia, Cerebral palsy, Neuromotor delay, Epilepsy * Correspondence: cfons@hsjdbcn.org; pedro.lazo@csic.es 6 Neurology Department, Hospital Sant Joan de Déu, Sant Joan de Déu Research Institute, Esplugues de Llobregat, Barcelona and CIBERER, Instituto de Salud Carlos III, Barcelona, Spain 1 Molecular Mechanisms of Cancer Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, Spain Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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The identification of underlying genetic al- Children with severe non-hereditary neurodevelopmen- terations in neuromotor developmental disorders is tal delay, and neonatal or early onset of epileptic sei- proving very useful in a significant number of neuro- zures, recently named as developmental and epileptic logical phenotypes [14–16]. encephalopathy [1, 2], frequently have an unknown eti- Human normal diversity is a consequence of different ology, which are likely to be very heterogeneous. In this combinations of non-pathogenic gene variants. Therefore, context, genetic alterations can play a relevant patho- it is likely that heterogeneity in neurological phenotypes genic role. Genomic studies based on whole-exome se- can also be a consequence of alternative combinations of quencing (WES) and single-nucleotide polymorphism genetic changes in genes associated to cellular functions (SNP) microarray to detect CNVs can identify genetic al- that are involved in the phenotype and constitute the indi- terations contributing to the pathogenic mechanism of vidual variome [8, 9]. Therefore, we tested this hypothesis these complex clinical phenotypes [3]. These underlying by searching for CNV variome differences among the four genetic alterations may be of different types, and the bet- family members of a complex case of a child, as test case, ter known are those associated to dominant point muta- that presented a developmental and epileptic encephalop- tions. However, the genetic heterogeneity of complex athy of neonatal onset that has a novel KCNQ2 mutation neurological diseases such as cerebral palsy [4], epilepsy resulting from a partial KCNQ2 exon 7 duplication that [5], and autism [6, 7] has already been associated to a impairs its inhibitory signal [17] and to determine if the combination of several different pathogenic gene vari- proband has a CNV pattern associated to neuronal func- ants, and to several CNVs in affected individuals, and tions. However, the complexity of the neurological syn- the combination of CNV changes contributes to the in- drome suggested that, although this mutation is necessary, dividual variome [8, 9]. CNVs are structural gene alter- there has to be additional cooperating genetic alterations. ations deleting or duplicating gene segments. Genomic We have approached the identification of the underlying SNP microarray studies have detected multiple CNV genetic problem by studying CNV alterations and their as- changes, deletions, and duplications, which are consist- sociation to genes whose function can contribute to the ent with the heterogeneity observed in epileptic patients neuropathological phenotype reported in the proband and [10], but no common pattern was identified. The pres- absent in the family [17], as an alternative approach to ence of a CNV in a gene can either alter its level of ex- searching for common variants among unrelated individ- pression, the stability of the RNA, the structure of the uals with similar phenotypes. Complex neurodevelopmen- protein, or its subcellular localization. Any of these ef- tal delay and epileptic phenotypes might be the result of fects will alter their function, which may be even more de novo combination of CNV alterations in genes associ- relevant in proteins associated to the membranes where ated to neurological functions that contribute to the pa- they can play a role in specific neural functions, ion and tient syndrome and determine its pathogenic mechanism. neurotransmitter transport, neural cell interactions, or signal transmission. Materials and methods Neonatal onset epilepsies related to KCNQ2 mutations Standard protocol approvals, registrations, and patient share a loss of the potassium channel function. However, consents a common loss of function can cause very heterogeneous The genomic studies were performed for diagnosis of a neurological phenotypes that accompany the epileptic pediatric neurological syndrome of unknown etiology. phenotype [11, 12]. Therefore, it is likely that there are For the diagnostic genomic studies, total DNA was ob- additional unidentified genetic changes that can contrib- tained from peripheral blood samples. Written informed ute to the heterogeneity and complexity of the individual consent for the genomic study was obtained from both neurological phenotype [5, 10, 12]. An alternative ap- parents. Research protocol and consent forms were ap- proach to detect candidate pathogenic genes is to search proved by the Institutional Review Boards of Hospital for specific CNV alterations in a proband with respect to Universitario de Salamanca-Centro de Investigación del the family members. Moreover, the analysis of CNVs in Cancer as reported in a previous publication [17]. these patients has detected a variable number of add- itional genetic changes, but did not identify a specific or SNP microarray unique pattern of specific gene alterations common to The SNP microarray analysis of genomic alteration was different patients [13]. In neurons, where ion channels performed using the matrix chip CytoScan HD array and specific cellular interactions are critical for its devel- (Affymetrix; Thermo Fisher Scientific, Inc.) following opment and correct functions, CNVs can alter protein manufacturer’s instructions. The matrix contains level, and their subcellular localization or density. These 2.696.550 probes that include 743,304 SNPs and 1,953, CNVs can play a pathogenic role if associated to the 246 non-polymorphic probes. The mean spacing be- neurological functions that are altered in the clinical tween probes for RefSeq genes is 800 bp, and 96% of the García-Hernández et al. Human Genomics (2021) 15:11 Page 3 of 11 genes are represented. Briefly, 500 ng of DNA was Genomics Viewer (IGV) [21] was used to inspect the se- digested with Nsp1 for 2 h at 37 °C. Digested DNA was quencing data and construct the graphs associated to purified and ligated to primers/adaptors at 16 °C for 15 WES-CNV analysis. h. The products of this ligation were used to generate amplicons by PCR using the primers provided by the Analysis of gene alterations and function associated to manufacturer (Affymetrix). The PCR program was one clinical phenotypes cycle at 94 °C for 1 min, thirty-five cycles (94 °C for 30 s, All genes alterations or variants detected by either SNP 60 °C for 45 s, and 65 °C for 1 min) and a final extension microarray or WES were analyzed using the VarElect cycle at 68ªC for 7 min. Purified PCR products were program [22] (LifeMap Sciences Inc., Tel Aviv, Israel), to digested with DNase I for 35 min at 37ªC, and the frag- search for a correlations between the biological function mented DNA was labeled with biotinylated nucleotides of altered genes and the clinical phenotypes of the case, with deoxynucleotide terminal transferase for 4 h at in order to detect a mechanistic implication of the gene 37ªC. A 250 μg of fragmented DNA was hybridized to contribution to clinical symptoms. the Chip Affymetrix chip Cytoscan HD preequilibrated at 50 °C for 18 h. The matrixes were washed and stained in the GeneChip Fluidics Station 450 (Affymetrix Inc.), Results and DAT images were acquired with the GeneChip Neuromotor developmental delay phenotype of patient Scanner 3000 7G (Affymetrix Inc.). The data files (archi- The patient is a 6-year-old boy presenting a cerebral ves.cel) were generated with the Affymetrix GeneChip palsy-like syndrome associated to severe development Command Console Software (AGCC) software (Affime- delay of unknown origin. The patient has a severe axial trix Inc., Santa Clara, CA). Data analysis was performed hypotonia without head control, spastic-dystonic tetra- using the analysis program package from Affymetrix paresis, and peripheral neuro-axonal motor neuropathy, Chromosome Analysis Suite (CHAS 4.0). The CGH re- hypertonia of all limbs with dystonic movements of sults were compared with data in ChAs 3.3 NetAffix arms, no hand use and is not able to sit or crawl, and is Build 33.2(Hg19) as reference. In addition, the aCGH re- unable to talk. He also presents an epileptic encephalop- sults were compared among the four members of the athy of neonatal onset with seizures well controlled since family to confirm their presence in the proband, but not 4 years of age [17]. The full clinical study has already in the parents and sister. Furthermore, the exome assay been reported [17]. The WES study detected a partial segments were determined through the Control-Freec KCNQ2 exon 7 duplication (Clinvar ID 617505) that im- program [18], using as reference either the father, pairs its function [17], but it did not identify any other mother, and daughter exomes. An example that that neuropathogenic mutation or gene variant [17] that shows the overlapping alterations (nCN-LOH, gains, and could be functionally associated to the complex neuro- losses) between the aCGH microarray and WES exome logical phenotype of this patient. assays for the patient with respect to the other three family members is represented in Supplementary Figure Cooperating CNVs in neuro-pathogenic genes S1. Graphs were depicted through the custom table op- We hypothesized that the proband might have additional tion in the UCSC Genome Browser for the GRCh37 hu- candidate genetic alterations, which must occur in genes man genome version. associated to neurological functions, and if they are known to have mutations, these mutants should also be Whole exome sequence and comparison with SNP associated to neurological phenotypes. Therefore, the microarray phenotypic convergence is due to a combination of sev- The WES study has been previously published in the re- eral alterations in genes whose protein biological func- port with the KCNQ2 exon 7 partial duplication [17]. tions can contribute to different aspects of this complex CN variant analysis in WES data was performed using neurological syndrome. In this context, we reasoned that VarScan2 [19]. additional genetic factors have to contribute to this com- Briefly, raw FASTQ files were mapped against the plex clinical phenotype, either in the form of additional hg19 version of the human genome using the BWA- genetic mutations or changes in gene copy number that MEM aligner. The resulting BAM files were pre- will alter the expression and level of their proteins. Be- processed following the GATK [20]: workflow, marking cause the WES study did not identify any additional PCR duplicates, and correcting errors in the base quality gene variant or mutant associated to the pathology, in scores. The exome assay CNV segments were deter- addition to the known KCNQ2 exon 7 partial duplica- mined through both the Control-Freec [18] and the tion [17], and in order to detect additional cooperating VarScan2 [19] programs, using as a reference either the gene alterations that contribute to the pathogenesis of father, mother, or daughter exomes. The Integrative the patient complex neurological syndrome, the genome García-Hernández et al. Human Genomics (2021) 15:11 Page 4 of 11 of the proband and family members was further studied genomic regions was determined by their previous asso- by SNP microarrays to detect CNVs. ciation of their known mutations to a neurological The SNP microarray study of the CNV variome in the phenotype. All the genes identified in the patient within patient can detect changes genes related to the altered CNVs that have a correlation with the clinical phenotype neurological functions, and therefore, genes located in are expressed in neurons, have specific functions associ- them are candidates to be involved in the clinical pheno- ated to the nervous system, or its known mutations have type. The number of CNVs was similar in the four mem- been associated to a neurological phenotype (Table 2). bers of the family (Table 1), most of them were already Functionally, most of these genes codify for several ion known, and a small proportion of them were de novo channel proteins or membrane proteins implicated in CNVs; however, they were not shared among the four neuronal cell interactions, which can affect synaptic family members. The microarray study detected in the transmission and cell polarization. This indicates that in- proband, compared to the other three family members, dividually these genes by themselves are not sufficient to several genomic de novo CNVs larger than 3 kb (Table 2), cause the phenotype, but can contribute to the disease and its markers are detailed in the Supplementary Table when they are combined with other genetic alterations S1. Larger de novo loss of heterozygosity (LOH) genomic in a unique individual. The affected boy presents a com- regions in the patient are detailed in Supplementary Table bined haploinsuficiency, mainly of CNV losses, that are S2. The genes included within these de novo CNVs likely to contribute to the pathogenic mechanism and unique to the proband child are indicated in Supplemen- the severity of the syndrome based on the expression tary Tables S1 and S2. In five genes, there is a deletion af- level and subcellular localization of these proteins, the fecting several exons (SLC17A5, RYR3, ATP1A2, ELAVL2, neurological functions associated to these proteins, and ANO3), one gene (PCDH19) has an exon duplication, to the neurological pathogenic phenotypes associated to three genes have intronic deletions (NALCN, CDH13, their genetic mutations in other patients. LRCC55), and two genes have an intronic duplication One gene implicated in neurotransmission, SLC17A5, (EFNA5, PCDH9). Intronic alterations can alter the pro- has a CNV loss that affects exons 7 to 9 (Table 3, Fig. 1). cessing of the RNA or its stability. The SLC17A5 protein is required for the transport of as- Next, we performed a search for a pathogenic associ- partate and glutamate into synaptic vesicles, which are ation of all the genes comprised within these CNVs de- driven by the membrane potential [30]. However, it is tected in the patient, by either SNP microarray or WES, not known whether a SLC17A5 haploinsuficiency, ex- and correlated their functions with different aspects of pressing, and aberrant protein might behave in a manner the clinical phenotype [17]. For this aim, the VarElect similar to its mutants regarding symptoms such as dys- program was used [22]. All changes in the proband were tonia, hypotonia, or seizure crisis and share some symp- also normalized with respect to the genome of the other toms such as hypotonia, ataxia, epilepsy, nystagmus, and family members, both parents and sister. The search was findings of cerebral and cerebellar atrophy detected in performed to identify functional and mechanistic corre- patients with Salla disease [33]. lations between gene functions and components of the RYR3 (ryanodine receptor type 3) codes for a pre- clinical phenotype such as seizures, dystonia, epilepsy, synaptic endoplasmic reticulum ryanodine receptor- neurotransmission, and motor neuron function. The mediated Ca2+ and forms a voltage-independent, nonse- neuropathogenicity of candidate genes within these lective, non-inactivating cation channel permeable to Table 1 Genetic changes detected by aCGH in the family. The complete list of CNVs detected in the patient are shown in supplementary Table S2 including its markers (rs identifiers), size, and chromosomal position. The CGH results were compared with data in ChAs 3.3 NetAffix Build 33.2(Hg19) as reference. Known means that they have already been detected in other unrelated individuals De novo CNV alterations in each family member Small CNV (1-3kbp) Large CNV CNV losses (autosomes/sex chrom) CNV gains (autosomes/sex chrom) losses (1–2 Mbp) Number Previously known New “de novo” Number Previously known New “de novo” Number Father 221 190/31 18/6 142 23/119 1/36 89 Mother 316 272/44 39/9 209 33/176 6/53 105 Daughter 352 261/92 34/25 421 68/353 5/108 106 Son (patient) 260 228/32 31/6 177 28/149 28/149 95 García-Hernández et al. Human Genomics (2021) 15:11 Page 5 of 11 Table 2 Genes with de novo CNV alterations in the proband that are functionally associated to neurological phenotypes GENE Chromosome Protein Protein functions Associated neuropathology (OMIM) CNV losses ATP1A2 1q23.2 ATPase Na+/K+ Na+/K+ ATPase maintains electrochemical gradient Rare forms of epilepsy and seizures [25–29] (182340) Transporting for electrical excitability of nerve and muscle. Subunit Alpha 2 Participates in neurotransmitter uptake and muscle contraction [23, 24] SLC17A5 6q13 Solute Carrier Affects membrane potential-driven aspartate and glu- Pathogenic variants or CNV loss of one allele (604322) Family 17 tamate transport into synaptic vesicles [30–32] Re- associated to hypotonia, ataxia, epilepsy, Member 5 quired for normal CNS myelination [33, 34]. seizures, nystagmus and findings of cerebral and cerebellar atrophy [33] ELAVL2 9p21.3 ELAV Like RNA Neural-specific RNA-binding protein that binds to sev- Alterations in neuronal differentiation and (601673) Binding Protein eral 3' UTRs. Expressed in early neuronal progenitors regulates neurodevelopmental and synaptic 2 to mature neurons [35] and required for normal neur- gene networks [36] onal development in the embryonic CNS LRRC55 11q21.32 Leucine Rich Subunit modulating gating properties in Ca2+- Altered neuronal polarization and (615213) Repeat activated potassium channel BK, and its voltage depolarization [37–40] Containing 55 dependence in the hyperpolarizing direction [37–40] PCDH9 13q21.32 Protocadherin 9 Ca2+-dependent cell adhesion in neural tissues. Epileptic encephalopathy [43] (603581) Protein involved in signaling at neuronal synaptic junctions [41, 42] NALCN 13q33.1 Sodium Leak Voltage-gated Na+ and Ca2+ channels regulating the NALCN deficiency is associated to (611549) Channel, Non- resting membrane potential and excitability of channelopathies [47]. NALCN pathogenic Selective neurons [44–46] variants associated to Neuroaxonal Dystrophy (INAD) patients, severe hypotonia, speech impairment, cognitive delay, epilepsy and mental disability [44, 45, 48, 49] NALCN associated to dystonia [46, 50] RYR3 15q14 Ryanodine Presynaptic endoplasmic reticulum ryanodine RYR3 haploinsuficiency cooperates SCN1A, (180903) Receptor 3 receptor-mediated Ca2+ release [51, 52]. Involved in implicated in epileps y[54, 55]. skeletal muscle contraction by releasing calcium from the sarcoplasmic reticulum followed by depolarization of T-tubules Regulates composition of the protein complex that forms a voltage-independent, nonselective, non- inactivating cation channel permeable to Na+, K+, and Ca2+, which regulates the neuronal background so- dium leak conductance [53] CDH13 16q23.3 Cadherin 13 Negative regulator of axon growth during neural Epileptic encephalopathy [43] (601364) differentiation [41, 43] ANO3 11p14.3 Anoctamine 3 Mutation in this gene exhibited abnormalities in ANO3 pathogenic variants have a dominant (610110) endoplasmic reticulum-dependent calcium signaling, effect on dystonia [58, 59] and to complex AbnormalCa2+-activated chloride channel [56, 57] neurological syndrome combining dystonia and myoclonus phenotypes [60, 61] CNV gains EFNA5 5q21.3 Ephrin A5 Involved in short-range contact-mediated axonal (601535) guidance. Prevents axon bundling of cortical neurons with astrocytes [62] PCDH19 Xq22.1 Protocadherin Calcium-dependent cell-adhesion protein primarily PCDH19 pathogenic variants and CNV in (300460) 19 expressed in the developing brain [63, 64] epilepsy [63–69] Na+, K+, and Ca2+, which regulates the neuronal back- regulate the intracellular calcium homeostasis that is al- ground sodium leak conductance [70]. Functionally, tered in neurodegenerative diseases and can play an im- RyRs proteins regulate the generation of plateau poten- portant role in the pathogenesis of motor neuron tials in motor neurons and also affect vesicle disorders (MND) [71]. The patient has a CNV loss that mobilization and synaptic plasticity [70]. In motor neu- includes exon 2 (Table 3, Supplementary Fig. S2). Fur- rons, the RYR3 protein regulates intracellular calcium, in thermore, haploinsuficiency of RYR3 might cooperate in which AMPA-type GluR (glutamate receptor) channels an indirect way with several membrane proteins coded García-Hernández et al. Human Genomics (2021) 15:11 Page 6 of 11 Table 3 Genes with exons altered by deletion or duplication Gene Exons deleted Exons duplicated CDS location of exons in gene sequence and aminoacids NCBI Ref. sequence PCDH19 3 59570-59628 (59 aa) NG_021319.1 SLC17A5 7 32031-32189 (159 aa) NG_008272.1 8 38546-38678 (133 aa) 9 43446-43593 (148 aa) RYR3 2 162453-162572 (120 aa) NG_047076.1 ELAVL2 3 95182-95285 (104 aa) NG_016425.2 ATP1A2 2 5177-5281 (105 aa) NG_008014.1 3 5463-5522 (60 aa) 4 7484-7687 (204 aa) 5 8214-8327 (114 aa) 6 8567-8701 (135 aa) 7 9407-9524 (117 aa) ANO3 3 252796-252990 (195 aa) NG_042856.1 4 254643-254714 (72 aa) 5 273908-274026 (119 aa) SLC17A5 Father Mother Daughter Son (Proband) Deleted region transcription SCA15A5 gene 11 10 9 8 7 654 3 2 1 exons Fig. 1 Exon deletion in SLC17A5 gene in the proband. The genomic region comprising the SLC17A5 gene in the four family members is shown. The region deleted in the proband is marked by a box and a red line. The arrow indicates the direction of transcription García-Hernández et al. Human Genomics (2021) 15:11 Page 7 of 11 by genes implicated in sodium or calcium voltage chan- extracellular domain of the protein, have been associated nels, including SCN1A that is also implicated in epilepsy with epilepsy, mostly in females, and recently were also [54]. detected in males [65]. Truncation mutations in The NALCN protein is a sodium leak channel [72] PCDH19 have also been associated to seizures [76]. expressed in neurons of the substantia nigra, and its re- PCDH19 is located on chromosome X and the patient duction impairs the spontaneous firing required for the has a CNV duplication that includes exon 3 (Table 3, inhibition of downstream brain areas [73]. The NALCN Supp. Fig. S5) that might alter its gene expression and protein interacts with UNC80 and pathogenic variants in protein stability or level and modify local interactions both genes have been associated to dystonia [44, 74]. among neural cells. CDH13 is a negative regulator of NALCN pathogenic variants have been associated to axon growth during neural differentiation [43], and a Neuroaxonal Dystrophy (INAD) patients, as well as to change in its protein level can also alter neuronal inter- patients with severe hypotonia, speech impairment, cog- actions and network organization. Both CDH13 and nitive delay, epilepsy, and mental disability [48]. The pa- PCDH9 have an intronic deletion and a duplication, re- tient has a CNV loss, an intronic deletion in NALCN spectively, and both have been associated to epileptic that alters its mRNA. Therefore, a reduction in the encephalopathy [75]. Some of these cadherins are NALCN protein level might mimic a defective NALCN- expressed in other organs such as the colon, kidney, UNC80 complex in the pathogenesis of dystonia. NALC heart, liver, and lung. However, the function of all these N deficiency has been associated to channelopathies and organs was normal in the proband. cervical dystonia [47]. Additional genes with CNV changes that can contrib- The SNP microarray analysis detected a large deletion ute to epileptic crisis and seizures were detected. that contains the ANO3 (Anoctamine 3) gene coding for LRRC55 (Leucine Rich Repeat Containing or BK Chan- a protein belonging to the TMEM16 family that func- nel Auxiliary Gamma Subunit) has a CNV loss deleting tions as a Ca (2+)-activated chloride channel. ANO3 the 3′ untranslated region of the mRNA in the patient. pathogenic variants have a dominant effect on dystonia LRRC55 gene is expressed in the cortex, cerebellum, and [58], and some have been associated to a complex spinal cord, and its protein is a regulator of large- neurological syndrome combining dystonia and myoclo- conductance, voltage, and calcium-activated potassium nus phenotypes [60]. In the proband, ANO3 has an LOH channel (BK alpha) that modulates its gating properties that incudes exons 3 to 5 (Table 3, Supplementary Fig. [37]. Also there is a deletion of exon 3 in the ELAVL2 S3), and its combination with pathogenic variants and gene (Supplementary Figure S6) coding for a neural- CVNs in other genes is likely to contribute to the specific RNA-binding protein that binds to several 3′ syndrome. UTRs and is expressed in early neuronal progenitors to Genes implicated in seizures and epileptic-like pheno- mature neurons [35] and also regulates co-expression types present alterations in EEG patterns. The CNV ana- networks of neurodevelopmental and synaptic genes lysis identified four genes with de novo alterations, three [36]. The EFNA5 coding for ephrin5A also has an in- with loses (ATP1A2, SLC17A5, and NALCN) and one tronic duplication that can affect its transcription or with a gain (PCDH19), which have a direct relation with RNA stability. Ephrin5 is implicated in neurodevelop- epileptic-like phenotypes (Table 1). ATP1A2 is highly ment and axon bundling [62]. expressed in the brain and codes for an integral mem- brane protein responsible for establishing and maintain- Discussion ing the electrochemical gradients of Na and K ions In patients with neurodevelopmental problems, there is across the plasma membrane. ATP1A2 has been associ- a large genetic heterogeneity implicating several regions ated, in several studies, to rare forms of epilepsy and sei- that present CNV changes. CNVs indicate a change in zures [25]. The proband has a loss comprising exons 2 gene dosage, which pathogenically means that there is a to 7 (Table 3, Supplementary Fig. S4). Other genes with lower, or higher, level of the protein in cells, depending CNV losses that have an indirect relation to epilepsy are on whether the type of change is a loss or a gain. Cellu- RYR3, CDH13, PCDH9, and LRRC55 (Table 2). lar functions are performed by proteins, and therefore, Three of the affected genes in the proband, PCDH19, their levels and subcellular distribution are an important PCDH9, and CDH13, code for members of the cadherin factor. The nervous system membrane-associated pro- protein family, which are implicated in neural intercellu- teins, such as those that participate in ion and neuro- lar interactions. PCDH19 mediates cell adhesion in transmitter channels, or in cell-cell interactions, are very neural tissues and regulates signaling at synaptic junc- likely to be affected depending of their level of expres- tions. Pathogenic PCDH19 variants and CNV changes sion and alteration in the number of functional com- affect this gene in epilepsy [75]. More than one hundred plexes in the cell. Functionally, a change in protein levels different PCDH19 pathogenic variants, located in the is a mechanism different from mutant proteins. The García-Hernández et al. Human Genomics (2021) 15:11 Page 8 of 11 change in protein level can affect its distribution and lo- consequently are likely contributors to alterations in EEG, cation in neuronal cell surface, leading to the alteration seizures, neurotransmission, epileptic-like crisis, or dys- of complexes in which they participate. This is particu- tonia. Moreover, protocadherins and cadherin CNVs by larly relevant for membrane proteins that can affect cel- altering their protein level and distribution on the cell sur- lular interactions, ion transport, or vesicle release, such face can affect neural cell-cell interactions. Therefore, it is as in synapsis. All these roles are essential for a normal not possible to attribute a single phenotype to any individ- neural development and its associated functions. ual gene, but the combination of several haplo-insufficient The clinical heterogeneity reflects a very complex situ- genes can generate a pathological situation related to their ation in which an unknown combination of alterations function. In this particular case, it is striking that four of in gene dosage and pathogenic variants is likely to con- the genes with a haploinsuficiency (LRRC5, NALCN, tribute to the pathogenesis of complex neurological phe- RYR3, and ANO3) affect calcium channels. CNVs in one notypes. However, for a clinical phenotype, the of the implicated genes, PCDH19 [75, 78], were already cooperation of genes related to that particular phenotype known to contribute to epileptic and neurodevelopmental are necessary, but the specific gene combination may delay syndromes [75]. vary from case to case and result in a neurological path- The simultaneous alterations of several gene coding way perturbation that is pathogenic [77]. for different ion channels and cell-cell interaction pro- The contribution of individual genes with CNV changes teins create a compound genetic haplo-insufficiency, to the patient phenotype is not known, but most of them which due to its complexity is likely to occur in patients occur in genes coding for proteins that participate in ion presenting complex neurodevelopmental delays with an channels or neural cell interactions, which can regulate unknown etiology. What is relevant regarding the af- ion transport and neurotransmission. Thus, it is likely that fected genes is their functional pathogenic combination an imbalance among several of these proteins and their rather than the implication of specific individual genes. functions contribute to complex neurological phenotypes. It is important to consider that these neurodevelopmen- In the case of this patient, functions of the OMIM genes tal phenotypes are likely to be the consequence of a identified in the CNV analysis cluster by their mechanisms complex pattern of alteration in gene dosage and expres- of action with the functional characteristics of the clinical sion levels of their proteins, rather that resulting from a phenotype (Fig. 2). The detection that several of the al- unique monogenic defect, as it occurs with human vari- tered genes, directly or indirectly, are implicated in ionic ation in multiple phenotypes, normal or pathological. (calcium, sodium) or glutamate transport and However, these complex clinical phenotypes can also be Neuronal cell Motorneuron Neuronal interactions function Ion channels membrane proteins PCDH19 NALCN membrane proteins dystonia ANO3 CDH13 seizures ATP1A2 PCDH9 SLC17A5 EFNA5 epilepsy RYR3 LRRC55 ELAVL2 KCNQ2dup Neural development Axon guidance Cell polarization / depolarization Neurotransmission Fig. 2 Pathogenic convergence between de novo altered genes and the clinical phenotype. The diagram illustrates the connection between OMIM genes with a pathogenic variant or CNV change that has a functional connection with the clinical phenotype of the patient. The lines indicate a direct association between a gene alteration (CNV or pathogenic variant) and the phenotype that has also been individually reported in other patients. The phenotypic associations were detected using the VarElect program to identify functional relations between genes and phenotypes. Dup: duplication García-Hernández et al. Human Genomics (2021) 15:11 Page 9 of 11 modulated by additional gene variants. It is likely that Additional file 7: Supplementary Table S1. de novo CNVs (> 1Kb) the clinical phenotype and its evolution will be condi- identified in patient. tioned by additional genetic alterations. Therefore, it is Additional file 8: Supplementary Table S2. Regions with loss of likely that alternative gene combinations can also cause heterozygosity (LOH)> 3 Mb in the patient. related and similar clinical phenotypes. Nowadays, the cause of cerebral palsy is changing from Abbreviations its origin in childbirth problems to an unknown origin, PCDH19: Protocadherin 19; CDH13: Cadherin 13; PCDH9: Protocadherin 9; ELAVL2: ELAV-like neuron-specific RNA binding protein 2; NALCN: Na+ leak which is likely to be genetic and heterogeneous. It is channel; ANO3: Ca2+-activated Cl- channel; ATP1A2: ATPase Na+/K+ possible that when unraveled, they might share common transporting subunit alpha 2; SLC17A5: Sodium-dependent Asp/Glu pathogenic pathways, although they might involve differ- transporter; RYR3: Brain ryanodine receptor-calcium release channel; LRRC55: Auxiliary protein of the large-conductance, voltage, and Ca2+-acti- ent genes that affect the same functions. The identifica- vated K+ channel; EFNA5: Ephrin A5 tion of gene/protein networks associated to the clinical neurological phenotype can set the bases for designing Authors’ contributions novel therapeutic approaches to manage these patients JLGH and LAC performed and analyzed the SNP microarray. IM-A and P-GP performed whole exome sequence study. CS coordinated the clinical and minimize, or compensate, the functional conse- characterization of the patient. PAL coordinated and integrated genetic and quences of neuropathogenic gene combinations. neurological studies and wrote the manuscript. The author(s) read and ap- proved the final manuscript. Conclusion Funding We conclude that the genetic heterogeneity of early severe Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e neurodevelopmental delays, cerebral palsy-like with dys- Innovación (SAF2016-75744-R, PID2019-105610RB-I00) and Consejería de Edu- cación-Junta de Castilla y León (CSI264P20, CLC-2017-01, and UIC-258) to tonia and epileptic encephalopathy, will have to be charac- P.A.L. Agencia Estatal de Investigación-Ministerio de Economía y terized in the context of the initiating pathogenic variant Competitividad-FEDER-Fondo Social Europeo (RTC-2017-6494-1 and RTI2018- that is modulated by several additional genetic changes 094434-B-I00) to P. G-P. Departament de Salut de la Generalitat de Catalunya-URDCat Project (SLT002/16/00174) to C.F. coding for proteins associated to neuronal functions. The study sponsors have no role in study design, in analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s40246-021-00309-4. Availability of data and materials SNP microarray data sets have been submitted to GEO with the identifier GSE122584. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122584 Additional file 1: Supplementary Figure S1. CNV from WES studies in Whole Exome Sequence (WES) raw data files are available in: the SLC17A5 gene. CNV alterations (Gains [red] and losses [blue]) https://www.ncbi.nlm.nih.gov/sra/PRJNA629061 retrieved by WES assays through the VarScan2 algorithm for the patient https://www.ncbi.nlm.nih.gov/bioproject/PRJNA629061 with respect to the other three family members. Graphs were depicted https://digital.csic.es/handle/10261/170405 using the integrative genome viewer (IGV) tool for the GRCh37 human genome version. Ethics approval and consent to participate Additional file 2: Supplementary Figure S2. Exon deletion in RYR3 All participants were provided with an explanation of the genetic study gene in the proband. The genomic region comprising the RYR3 gene in research, and written informed consent for the diagnostic genetic study was the four family members is shown. The region deleted in the proband is obtained from the parents of the affected child. The genomic diagnostic marked by a box and a red line. The arrow indicates the direction of study was approved by the ethics committee of the Hospital Universitario de transcription. Salamanca and Hospital Sant Joan de Deu. Additional file 3: Supplementary Figure S3. Exon deletion in ANO3 gene in the proband. The genomic region comprising the ANO3 gene in Consent for publication the four family members is shown. The region deleted in the proband is Written informed consent for publication was obtained from the participant’s marked by a box and a red line. The arrow indicates the direction of legal guardian/next of kin. transcription. Additional file 4: Supplementary Figure S4. Exon deletion in ATP1A2 Competing interests gene in the proband. The genomic region comprising the ATP1A2 gene The authors declare they have no competing interests. in the four family members is shown. The region deleted in the proband is marked by a box and a red line. The arrow indicates the direction of Author details transcription. 1 Molecular Mechanisms of Cancer Program, Instituto de Biología Molecular y Additional file 5: Supplementary Figure S5. Duplication Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), encompassing exon 3 in the PCDH19 gene. The genomic region Universidad de Salamanca, Salamanca, Spain. 2Instituto de Investigación comprising the PCDH19 gene in the four family members is shown. The Biomédica de Salamanca (IBSAL), Departamento de Hematología, Hospital region deleted in the proband is marked bya box and a red line. The Universitario de Salamanca, Salamanca, Spain. 3Network Center for arrow indicates the direction of transcription. Biomedical Research in Cancer (CIBERONC), Salamanca, Spain. 4Centro de Additional file 6: Supplementary Figure S6. Deletion encompassing Biología Molecular Severo Ochoa, CSIC-Universidad Autónoma de Madrid, exon 3 in the ELAVL2 gene. The genomic region comprising the ELAVL2 Cantoblanco, Madrid, Spain. 5Biosciences Research Institute, School of gene in the four family members is shown. The region deleted in the Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain. 6Neurology Department, Hospital Sant Joan de Déu, Sant Joan proband is marked by a box and a red line. The arrow indicates the direction of transcription. de Déu Research Institute, Esplugues de Llobregat, Barcelona and CIBERER, Instituto de Salud Carlos III, Barcelona, Spain. García-Hernández et al. Human Genomics (2021) 15:11 Page 10 of 11 Received: 17 November 2020 Accepted: 26 January 2021 19. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, Miller CA, Mardis ER, Ding L, Wilson RK. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 2012;22:568–76. https://doi.org/10.1101/gr.129684.111. References 20. 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