Neuromotor developmental delay phenotype of patient
The patient is a 6-year-old boy presenting a cerebral palsy-like syndrome associated to severe development delay of unknown origin. The patient has a severe axial hypotonia without head control, spastic-dystonic tetraparesis and peripheral neuro-axonal motor neuropathy, hypertonia of all limbs with dystonic movements of arms, no hand use and is not able to sit or crawl, and is unable to talk. He also present an epileptic encephalopathy of neonatal onset with seizures well controlled since 4 years of age [17]. The full clinical study has already been reported [17]. The WES study detected a partial KCNQ2 exon 7 duplication (Clinvar ID 617505) that impairs its function [17], but it did not identify any other neuropathogenic mutation or gene variant [17] that could be functionally associated to the complex neurological phenotype of this patient.
Cooperating CNVs in neuro-pathogenic genes
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 2). 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 3, 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].
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 3, Supplementary Fig. S2). 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 3, 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 3, Supplementary Fig. S4). Other genes with CNV losses that have an indirect relation to epilepsy are RYR3, CDH13, PCDH9 and LRRC55 (Table 2).
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 3, Supp. Fig. S5) 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]. Some of these cadherins are expressed in other organs such as colon, kidney, heart, liver and lung. However, the function of all these organs was normal in the proband.
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 S6) 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].