Cooperating CNVs in neuro-pathogenic genes
We hypothesized that the proband might have additional candidate genetic alterations, which must occur in genes associated to neurological functions, and if they are known to have mutations, these mutants should also be associated to neurological phenotypes. Therefore, the phenotypic convergence due to a combination of several alterations in genes whose protein biological functions can contribute to different aspects of this complex neurological syndrome. In this context, we reasoned that additional genetic factors have to contribute to this complex clinical phenotype, either in the form of additional genetic mutations or changes in gene copy number that will alter the expression and level of their proteins. Because the WES study did not identify any additional gene variant or mutant associated to the pathology, in addition to the known KCNQ2 exon 7 partial duplication [17], and in order to detect additional cooperating gene alterations that contribute to the pathogenesis of the patient complex neurological syndrome, the genome of the proband and family members was further studied by SNP microarrays to detect CNVs.
The SNP microarray study of the CNVs variome in the patient can detect changes genes related to the altered neurological functions, and therefore genes located in them are candidates to be involved in the clinical phenotype. The microarray study detected in the proband, compared to the other three family members, several genomic de novo CNVs larger than 3 Kb (Table 1), and its markers are detailed in the Supplementary Table S1. Larger loss of heterozygosity (LOH) genomic regions in the patient are detailed in Supplementary Table S2. The genes included within these CNVs are indicated in Supplementary Tables S1 and S2. In five genes there is a deletion affecting several exons (SLC17A5, RYR3, ATP1A2, ELAVL2, ANO3), one gene (PCDH19) has an exon duplication, three genes have intronic deletions (NALCN, CDH13, LRCC55), and two genes have an intronic duplication (EFNA5, PCDH9). Intronic alterations can alter the processing of the RNA or its stability.
Table 1
Genes with CNV alterations functionally associated to neurological phenotypes
GENE (OMIM) | Chromo some | Protein | Protein Functions | Associated Neuropathology |
CNV losses |
ATP1A2 (182340) | 1q23.2 | ATPase Na+/K + Transporting Subunit Alpha 2 | Na+/K + ATPase maintains electrochemical gradient for electrical excitability of nerve and muscle. Participates in neurotransmitter uptake and muscle contraction [47, 48] | Rare forms of epilepsy and seizures [36, 49–52] |
SLC17A5 (604322) | 6q13 | Solute Carrier Family 17 Member 5 | Affects membrane potential-driven aspartate and glutamate transport into synaptic vesicles [23], [53, 54] Required for normal CNS myelination [24, 55]. | Pathogenic variants or CNV loss of one allele associated to hypotonia, ataxia, epilepsy, seizures, nystagmus and findings of cerebral and cerebellar atrophy [24] |
ELAVL2 (601673) | 9p21.3 | ELAV Like RNA Binding Protein 2 | Neural-specific RNA-binding protein that binds to several 3' UTRs. Expressed in early neuronal progenitors to mature neurons [42] and required for normal neuronal development in the embryonic CNS | Alterations in neuronal differentiation and regulates neurodevelopmental and synaptic gene networks [43] |
LRRC55 (615213) | 11q21.32 | Leucine Rich Repeat Containing 55 | Subunit modulating gating properties in Ca2+- activated potassium channel BK, and its voltage dependence in the hyperpolarizing direction [41, 56–58] | Altered neuronal polarization and depolarization [41, 56–58] |
PCDH9 (603581) | 13q21.32 | Protocadherin 9 | Ca2+-dependent cell adhesion in neural tissues. Protein involved in signaling at neuronal synaptic junctions [59, 60] | Epileptic encephalopathy [40] |
NALCN (611549) | 13q33.1 | Sodium Leak Channel, Non-Selective | Voltage-gated Na+and Ca2+ channels regulating the resting membrane potential and excitability of neurons [30, 61, 62] | NALCN deficiency is associated to channelopathies [33]. NALCN pathogenic variants associated to Neuroaxonal Dystrophy (INAD) patients, severe hypotonia, speech impairment, cognitive delay, epilepsy and mental disability [30, 32, 61, 63] NALCN associated to dystonia [62, 64] |
RYR3 (180903) | 15q14 | Ryanodine Receptor 3 | Presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+ release [65] [66]. Involved in 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 sodium leak conductance [67] | RYR3 haploinsuficiency cooperates SCN1A, implicated in epilepsy.[27, 68] |
CDH13 (601364) | 16q23.3 | Cadherin 13 | Negative regulator of axon growth during neural differentiation [40, 59] | Epileptic encephalopathy [40] |
ANO3 (610110) | 11p14.3 | Anoctamine 3 | Mutation in this gene exhibited abnormalities in endoplasmic reticulum-dependent calcium signaling, AbnormalCa2+-activated chloride channel [69, 70] | ANO3 pathogenic variants have a dominant effect on dystonia [34, 71] and to complex neurological syndrome combining dystonia and myoclonus phenotypes [35, 72] |
CNV gains |
EFNA5 (601535) | 5q21.3 | Ephrin A5 | Involved in short-range contact-mediated axonal guidance. Prevents axon bundling of cortical neurons with astrocytes [44] | |
PCDH19 (300460) | Xq22.1 | Protocadherin 19 | Calcium-dependent cell-adhesion protein primarily expressed in the developing brain [73, 74] | PCDH19 pathogenic variants and CNV in epilepsy [38, 73–78] |
Next, we performed a search for a pathogenic association of all the genes comprised within these CNVs detected in the patient, by either SNP microarray or WES, and correlated their functions with different aspects of the clinical phenotype [17]. For this aim the VarElect program was used [22]. All changes in the proband were also normalized with respect to the genome of the other family members, both parents and sister. The search was performed to identify functional and mechanistic correlations between gene functions and components of the clinical phenotype such as seizures, dystonia, epilepsy, neurotransmission, and motor neuron function. The neuropathogenicity of candidate genes within these genomic regions was determined by their previous association of their known mutations to a neurological phenotype. All the genes identified in the patient within CNVs that have a correlation with the clinical phenotype are expressed in neurons, have specific functions associated to the nervous system, or its known mutations have been associated to a neurological phenotype (Table 1). Functionally, most of these genes codify for several ion channel proteins or membrane proteins implicated in neuronal cell interactions, which can affect synaptic transmission and cell polarization. This indicates that individually these genes by themselves are not sufficient to cause the phenotype, but can contribute to the disease when they are combined with other genetic alterations in a unique individual. The affected boy presents a combined haploinsuficiency, mainly of CNV losses, that are likely to contribute to the pathogenic mechanism and the severity of the syndrome based on the expression level and subcellular localization of these proteins, the neurological functions associated to these proteins, and to the neurological pathogenic phenotypes associated to their genetic mutations in other patients.
One gene implicated in neurotransmission, SLC17A5, has a CNV loss that affects exons 7 to 9 (Table 2, Fig. 1). The SLC17A5 protein is required for the transport of aspartate and glutamate into synaptic vesicles, which are driven by the membrane potential [23]. However, it is not known whether a SLC17A5 haploinsuficiency, expressing and aberrant protein, might behave in a manner similar to its mutants regarding symptoms such as dystonia, hypotonia or seizure crisis and share some symptoms such as hypotonia, ataxia, epilepsy, nystagmus and findings of cerebral and cerebellar atrophy detected in patients with Salla disease [24].
Table 2
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 8 9 | | 32031–32189 (159 aa) 38546–38678 (133 aa) 43446–43593 (148 aa) | NG_008272.1 |
RYR3 | 2 | | 162453–162572 (120 aa) | NG_047076.1 |
ELAVL2 | 3 | | 95182–95285 (104 aa) | NG_016425.2 |
ATP1A2 | 2 3 4 5 6 7 | | 5177–5281 (105 aa) 5463–5522 (60 aa) 7484–7687 (204 aa) 8214–8327 (114 aa) 8567–8701 (135 aa) 9407–9524 (117 aa) | NG_008014.1 |
ANO3 | 3 4 5 | | 252796–252990 (195 aa) 254643–254714 (72 aa) 273908–274026 (119 aa) | NG_042856.1 |
RYR3 (ryanodine receptor type 3) codes for a presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+, and forms a voltage-independent, nonselective, non-inactivating cation channel permeable to Na+, K+, and Ca2+, which regulates the neuronal background sodium leak conductance [25]. Functionally, RyRs proteins regulate the generation of plateau potentials in motor neurons and also affects vesicle mobilization and synaptic plasticity [25]. In motor neurons, the RYR3 protein regulates intracellular calcium, in which AMPA-type GluR (glutamate receptor) channels regulate the intracellular calcium homeostasis that is altered in neurodegenerative diseases and can play an important role in the pathogenesis of motor neuron disorders (MND) [26]. The patient has a CNV loss that includes exon 2 (Table 2, Supplementary Fig. S1). Furthermore, haploinsuficiency of RYR3 might cooperate in an indirect way with several membranes proteins coded by genes implicated in sodium or calcium voltage channels, including SCN1A that is also implicated in epilepsy [27].
The NALCN protein is a sodium leak channel [28] expressed in neurons of the substantia nigra, and its reduction impairs the spontaneous firing required for the inhibition of downstream brain areas [29]. The NALCN protein interacts with UNC80 and pathogenic variants in both genes have been associated to dystonia [30, 31]. NALCN pathogenic variants have been associated to Neuroaxonal Dystrophy (INAD) patients, as well as to patients with severe hypotonia, speech impairment, cognitive delay, epilepsy and mental disability [32]. The patient has a CNV loss, an intronic deletion in NALCN that alters its mRNA. Therefore, a reduction in the NALCN protein level might mimic a defective NALCN-UNC80 complex in the pathogenesis of dystonia. NALCN deficiency has been associated to channelopathies and cervical dystonia [33].
The SNP microarray analysis detected a large deletion that contains the ANO3 (Anoctamine 3) gene coding for a protein belonging to the TMEM16 family that functions as a Ca (2+)-activated chloride channel. ANO3 pathogenic variants have a dominant effect on dystonia [34], and some have been associated to a complex neurological syndrome combining dystonia and myoclonus phenotypes [35]. In the proband, ANO3 has an LOH that incudes exons 3 to 5 (Table 2, Supplementary Fig. S2), and its combination with pathogenic variants and CVNs in other genes is likely to contribute to the syndrome.
Genes implicated in seizures and epileptic-like phenotypes present alterations in EEG patterns. The CNV analysis identified four genes with de novo alterations, three with loses (ATP1A2, SLC17A5 and NALCN) and one with a gain (PCDH19), which have a direct relation with epileptic-like phenotypes (Table 1). ATP1A2 is highly expressed in brain and codes for an integral membrane protein responsible for establishing and maintaining the electrochemical gradients of Na and K ions across the plasma membrane. ATP1A2 has been associated, in several studies, to rare forms of epilepsy and seizures [36]. The proband has a loss comprising exons 2 to 7 (Table 2, Supplementary Fig. S3). Other genes with CNV losses that have an indirect relation to epilepsy are RYR3, CDH13, PCDH9 and LRRC55 (Table 1).
Three of the affected genes in the proband, PCDH19, PCDH9, and CDH13, code for members of the cadherin protein family, which are implicated in neural intercellular interactions. PCDH19 mediates cell adhesion in neural tissues and regulates signaling at synaptic junctions. Pathogenic PCDH19 variants and CNV changes affect this gene in epilepsy [37]. More than one hundred different PCDH19 pathogenic variants, located in the extracellular domain of the protein, have been associated with epilepsy, mostly in females, and recently were also detected in males [38]. Truncation mutations in PCDH19 have also been associated to seizures [39]. PCDH19 is located on chromosome X and the patient has a CNV duplication that includes exon 3 (Table 2, Supp. Fig. S4) that might alter its gene expression and protein stability or level, and modify local interactions among neural cells. CDH13 is a negative regulator of axon growth during neural differentiation [40] and a change in its protein level can also alter neuronal interactions and network organization. Both, CDH13 and PCDH9 have an intronic deletions and a duplication respectively, and both have been associated to epileptic encephalopathy [37].
Additional genes with CNV changes that can contribute to epileptic crisis and seizures were detected. LRRC55 (Leucine Rich Repeat Containing or BK Channel Auxiliary Gamma Subunit) has a CNV loss deleting the 3’ untranslated region of the mRNA in the patient. LRRC55 gene is expressed in the cortex, cerebellum and spinal cord, and its protein is a regulator of large-conductance, voltage and calcium-activated potassium channel (BK alpha), that modulates its gating properties [41]. Also there is a deletion of exon 3 in the ELAVL2 gene (Supplementary Figure S5) coding for a neural-specific RNA-binding protein that binds to several 3' UTRs and is expressed in early neuronal progenitors to mature neurons [42] and also regulates co-expression networks of neurodevelopmental and synaptic genes [43]. The EFNA5 coding for ephrin5A also has an intronic duplication that can affect its transcription or RNA stability. Ephrin5 is implicated in neurodevelopment and axon bundling [44].