Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function.

Introduction

Voltage-gated calcium channels (VGCCs) are a family of calcium ion selective proteins that both mediate calcium entry into neurons at synapses upon depolarization and are required for calcium-dependent functions in the cell [1, 2]. VGCCs are composed of multiple subunits, including the α1 subunit which is encoded by the CACNA1A gene and is responsible for Ca2+ entry in neurons. Across species the α1 subunit is required in the nervous system. In mice the tottering (tg) mutants have mutations in Cacna1a and exhibit ataxia, motor seizures and cerebellar degeneration [3]. In Drosophila the channel is required for synaptic transmission and cacophony (cac) null alleles are lethal [4, 5]. In mosaic clones mutations in cacophony produce neurodegeneration with defective lysosomal fusion with autophagosomes [6].

Given the essential role of VGCCs in the nervous system it is not surprising that mutations in CACNA1A cause a spectrum of neurological disorders in humans[7]. Spinocerebellar ataxia type 6 (SCA6, OMIM #183086) is characterized by ataxia, dysarthria, and dysphagia with onset typically in middle adulthood but ranging from 20 to 60 years of age[8]. SCA6 is due to heterozygous expansions of CAG repeats within CACNA1A resulting in large polyglutamine tracts within the protein which cause cytoplasmic aggregations and Purkinje cell degeneration [9, 10]. Episodic ataxia type 2 (EA2, OMIM #108500) is characterized by episodes of ataxia with onset in childhood or early adulthood, and affected individuals are often responsive to carbonic anhydrase inhibitors such as acetazolamide [11-13]. EA2 is due to heterozygous deletions, stop-gains, frameshifts, or missense mutations [11, 14, 15]. Functional analysis based on patch-clamping suggests that they correspond to loss of channel function and hence reveal haploinsufficiency of the locus [16, 17]. While EA2 is typically episodic, many patients also have progressive ataxia due to ongoing neurodegeneration which is generally slow [15]. Familial hemiplegic migraine (FHM, OMIM # 141500) is a form of migraine with aura and transient hemiplegia and an age of onset between 5 years and early adulthood [18]. CACNA1A variants associated with FHM are typically missense alleles [19], and in contrast to EA2-associated missense alleles, electrophysiological evidence suggests that they are gain-of-function mutations [20]. FHM mutations appear to lead to hyperactivity of the channel [21] both by altering the biophysical properties as well as decreasing the inhibitory G-protein association with the channel [22].

While the disease phenotypes for EA2 and FHM appear to relate to different underlying genetic mechanisms, considerable phenotypic overlap between FHM and EA2 means that some individuals exhibit features of both disorders [23, 24]. Indeed even within the same family, the same CACNA1A variants can produce phenotypes more similar to FHM than EA2 [23, 25]. Several de novo missense alleles in CACNA1A have been reported in children with congenital ataxia and intellectual disability [26], as well as with non-progressive congenital ataxia with seizures[27]. However, channel function and its relation to phenotype are not well studied in these severe ataxias [26-28]. Most previous studies used in vitro electrophysiological analyses to assess channel function [20, 21, 29], leaving a need for functional annotation of CACNA1A and its variants in a model organism.

Results

Human subjects

We ascertained 5 individuals who underwent exome sequencing for global developmental delay and congenital ataxia, in whom de novo missense variants in CACNA1A were discovered. Patient 1 was enrolled in the Undiagnosed Diseases Network (UDN) at Baylor College of Medicine and concurrently in the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) [30] as part of a large-scale research re-analysis of clinical exomes [31]. Patients 2–5 were identified at Baylor Genetics Laboratories (BGL) [32, 33]. All families gave written consent for exome sequencing.

The clinical findings of these individuals are summarized in . All individuals (5/5) exhibited global developmental delay, expressive language delay and dysarthric (4/5) or no expressive speech (1/5; Patient 1). Interestingly all subjects had ataxia (5/5) but to varying degrees, with independent ambulation and unsteady gait in some (3/5) and more severely impaired ambulation requiring use of walker in others (2/5). Other neurological features such as behavioral problems, sensory processing disorders, aggressive behavior and attention deficit were also noted, although these features were not consistent among the patients.

Neuroimaging for the patients differed; one of the five subjects had evidence of progressive cerebellar atrophy. In Patient 1, the initial MRI at 10 months showed a normal cerebellum (), while imaging at 22 months revealed mild cerebellar atrophy (), which progressed at 3.5 years () and 8 years (). Patient 1 is the only subject with progressive cerebellar degeneration. In Patient 3, the cerebellum appeared normal in size at 2 years, although the corpus callosum was thin posteriorly (). In Patient 5 the MRI is normal (). In Patient 4 there is some cerebellar hypoplasia involving the vermis () but not the lobes of the cerebellum ()

Neuroimaging characteristics of the patients with CACNA1A de novo variants.

A) A T1 sequence showing an approximately mid-sagittal image of Patient 1 at 10 months of age. The cerebellum appears normal in size. B) Patient 1, a 3D FFE image at 22 months again showing a mid-sagittal view, with some increased prominence of the cerebellar folia suggesting early cerebellar atrophy C)Patient 1, T1 mid-sagittal view at 3.5 years showing progressive cerebellar atrophy D)Patient 1, a mid-sagittal T1 3D image at 8 years showing the neurodegenerative progression affecting the cerebellum E) Patient 3, a mid-sagittal T1 image at age 2 years, with normal cerebellum and thin corpus callosum posteriorly F) Patient 5, a mid-sagittal T1 image at age 18 months, normal.

Whole exome sequencing

Trio-based exome sequencing for Patient 1 through BHCMG [31] revealed a de novo missense variant (NM_001127221:c.5018G>C: p.R1673P; chr19:13346480C>G [hg19]) in CACNA1A (). Patients 2–5 had clinical proband exome sequencing at BGL [32, 33] (see Materials and Methods) and were all found to have a recurrent missense de novo CACNA1A variant (NM_001127221:c.4991G>A: p.R1664Q; chr19:13346507C>T [hg19]) (). Notably, the four individuals with p.R1664Q and the single individual with p.R1673P all exhibit some similarities to a child previously reported with a de novo p.R1664Q allele with early onset ataxia without seizures or migraine[34].

For each case the ratio of variant reads to total reads and the Sanger confirmation suggested approximately 50% variant alleles meaning the patients are heterozygous. Both de novo changes occur within CpG dinucleotides, hypermutable sites prone to methylation and deamination leading to de novo events [35-37]. These have been noted to affect arginine residues in a number of disease contexts [38-40]. The de novo missense changes in these patients affect conserved arginine residues at the S4 transmembrane segment of domain IV of the protein (). Several pathogenic alleles associated with a range of phenotypes are reported to affect this transmembrane segment of domain IV in ClinVar [41] ( For example, p.R1661H is associated with EA2, while p.R1667W is associated with FHM. The R1664Q allele seen in our patients 2–5 is associated with ataxia and global developmental delay. We observed that the intervals of the transmembrane domains (I-IV) all appear intolerant to missense variation ().

CACNA1A de novo variants affecting the S4 transmembrane segment of domain IV.

A graphical representation of the overall structure of the α1 subunit of the voltage gated calcium channel encoded by CACNA1A. A) Both the R1673P (red star) (Patient 1) and R1664Q (green star) (Patients 2–5) variants occur in the S4 transmembrane segment of domain IV. Missense alleles from ClinVar in the S4 transmembrane segment of domain IV are shown as black stars. Missense changes in this segment appear associated with Episodic Ataxia 2 (p.R1661H), familial hemiplegic migraine (p.R1667W) and the severe congenital ataxia in patients in our series (p.R1664Q and p.R1673P). The p.R1664Q (Patients 2–5) is listed in ClinVar as associated with SCA type 6, chronic and progressive ataxia and global developmental delay. While the p.R1673P variant is novel, ClinVar does list p.R1673C as a variant of uncertain significance. B) Minor allele frequency for missense variants listed in the ExAC browser plotted according to the codon position. Only alleles with MAF <0.01 are plotted. Missense alleles appear rarely or with very low allele frequency within the four transmembrane domains of the CACNA1A protein. CACNA1A is intolerant to loss-of-function variation with 4 such variants observed in ExAC over 78.3 expected (probability of loss-of-function intolerance = 1.0)[53]. Furthermore, the pattern of missense variation in ExAC across the coding region of CACNA1A is consistent with paucity of variation within the transmembrane segments.

Drosophila cac mutants and Drosophila transgenics

Notably these S4 arginine variants in domain IV display diverse phenotypes, and other severe cases reported also carry missense variants within the S4 segments in domains III [26, 27, 42] and IV [34] of CACNA1A. However, these mutations have not been modeled in vivo, and the functions of these missense variants are therefore not defined (i.e., haploinsufficient loss or gain of function). As drugs are available to either boost or inhibit calcium channel function, knowledge of mutation mechanism may have important implications for patients. In Drosophila cacophony (cac), the homolog of CACNA1A, is required for synaptic transmission and lysosomal fusion, and cac null alleles are embryonic lethal [4, 5]. Previously, we isolated numerous alleles of cac in a forward genetic screen for essential genes that affect the function of photoreceptors based on defective ERGs in homozygous mutant eye clones [20]: cac is an early nonsense mutation which is an embryonic lethal, and cac is a missense mutation affecting a key glutamate residue in the calcium ion selectivity filter loop and is larval lethal (). In mosaic eye clones, both mutations lead to expanded nerve terminals, synaptic vesicle accumulations, and aberrant lysosome-autophagosome fusion defects [6].

Drosophila lethality rescue experiments testing the de novo CACNA1A variants affecting the S4 transmembrane segment of domain IV.

A) The Drosophila cac genomic context showing the P[acman] clone CH321-60D21. B) The membrane topology of the voltage gated channel showing the cac alleles used in this study and the position of the human variants that were affecting conserved residues engineered into transgenes. C) Protein alignment of CACNA1A and its homologs in mouse, Drosophila and zebrafish, showing the strong conservation of the entire S4 segment of domain IV. D) Lethality rescue experiments in Drosophila for cac lethal alleles by wild type, R1673P or R1664Q mutant genomic transgenes. Lethality rescue experiments in Drosophila were done by crossing y w, cac FRT19A/FM7c or y w, cac FRT19A/FM7c with y w/Y; Dp(1;3)DC131(or -R1673P or -R1664Q) flies. The males that have no FM7c marker in the next generation were considered rescued flies. The numbers of rescued flies were also compared with FM7c males in the same progeny. The rescue results are as follows: GR-WT for cac: Rescued males (115) / FM7c males (67) = 1.72. GR-WT for cac: Rescued males (90) / FM7c males (56) = 1.61. GR- R1673P for cac: Rescued males (0) / FM7c males (86) = 0. GR- R1673P for cac: Rescued males (0) / FM7c males (81) = 0. GR- R1664Q for cac: Rescued males (50) / FM7c males (71) = 0.71. GR- R1664Q for cac: Rescued males (41) / FM7c males (67) = 0.61. E) Electroretinograms of 3-day-old flies. An ERG trace consists of an amplitude (red bracket), an on-transient and an off-transient (red dotted circles). Quantifications of the on-transients and off-transients of ERG traces are on the right. Data are presented as mean ± SEM. p values were calculated using Student’s t test. ***p < 0.001; NS, not significant. p-values were consistent with those on one way ANOVA. In 3-day-old cac mutant clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) the electroretinogram has a normal pattern of synaptic activity (demonstrated by on- and off-transients) and depolarization (amplitude marker). In photoreceptors from cac mutants there is a loss of “on” and “off.” In the cac mutants with the mutant transgenes (GR-R1673P, GR-R1664Q) there are distinct findings with R1673P producing larger “on” and “off” transients while the R1664Q produces a loss-of-function phenotype similar to the cac mutants.

To test the functional consequences of the variants identified in the five subjects, we designed a rescue-based strategy. To rescue the phenotypes associated with the cac alleles, we first selected a 77 kb P[acman] transgenic construct [6] that contains the entire 53 kb genomic region of cac, including endogenous enhancers to drive proper expression of the transgene. We introduced the two variants found in the subjects by recombineering and we inserted these genomic rescue (GR) constructs into the identical VK37 docking site [6] in the fly genome by phiC31-mediated recombination [6] to avoid position effects (). We labeled these constructs GR-WT (fly wild type), GR-R1673P (Patient 1) and GR-R1664Q (Patients 2–5) corresponding to the human proteins/variants. These mutations are within the S4 transmembrane segment of domain IV that is nearly identical between flies and humans ().

The wild type P[acman] transgene rescues the lethality associated with Drosophila cac and cac ( However, GR-R1673P (Patient 1) mutation failed to rescue lethality, whereas GR-R1664Q (Patients 2–5) was able to rescue lethality partially (41% of expected viable progeny). This data suggest that the two mutations have functional consequences in vivo, and that the R1673P mutation seems to behave as a more severely impaired allele.

Electroretinograms reveals differential effects in CACNA1A alleles

Next we performed ERGs [6] in cac mutant clones rescued with either a wild type or a mutant P[acman] GR construct. ERG recordings reveal two key features: the ‘on’ and ‘off’ transients (red dotted circles, ) typically reflect synaptic transmission between pre- and post-synaptic cells, whereas the amplitude of the depolarization (red bracket in ) is a measure of the phototransduction activity. cac mutations typically affect the ‘on’ and ‘off’ transients but have little or no effect on the amplitude, consistent with the role of cac in synaptic vesicle release [6]. To assess the function of the variants from the subjects, we generated homozygous mutant eye clones of cac and cac in young animals (3 days old) and compared the ERGs in flies that carry the wild type and mutant P[acman] GR constructs. Both cac alleles exhibit loss of synaptic transmission as evidenced by loss of the ‘on’ and ‘off’ transients (). Mutant flies carrying the wild type P[acman] cac transgene exhibited normal ‘on’ and ‘off’ transients (). Interestingly, GR-R1673P (Patient 1) dramatically increased the amplitude of the on and off transients, whereas GR-R1664Q (Patients 2–5) failed to rescue the synaptic transmission defect caused by the cac and cac mutations. These data suggest that the R1673P allele seen in Patient 1 is a gain-of-function mutation, and R1664Q found in Patients 2–5 is a loss-of-function allele based on the ERG defects observed in the photoreceptors in Drosophila. We also examined the ‘on’ and ‘off’ transients of flies with the P[acman] transgenes in a wild-type y w (cac +/cac+) background as well as heterozygous backgrounds (cac /+ and cac /+). Although there was a slight nominal increase in the ‘on’ transient in the R1673P animals, it was not statistically significant (S2B Fig).

As increased Ca2+ influx typically causes excitotoxicity which may lead to the demise of neurons [6], we recorded the ERGs of 8 genotypes (cac or cac mutations rescued by GR-WT, GR-1673P, GR-1664Q, or no rescue) in 30-day-old flies. We observed a loss of depolarization amplitude in the R1673P (Patient 1) flies in the cac (missense) mutant background (). In contrast, GR-R1664Q (Patients 2–5) did not lead to a significant decrease in amplitude. Interestingly, when the transgenes were tested in the cac (nonsense) mutant background the reduction in amplitude for R1673P was less severe although still statistically significant (). Similar to those tested in cac background, the R1664Q variant did not lead to a significant decrease in amplitude (). These data suggest that the increase in activity observed in young flies that carry the R1673P variant leads to age-dependent deterioration of the phototransduction pathway, a phenotype that has not been previously observed in cac loss-of-function mutations [6].

Functional and ultrastructural neurodegeneration in CACNA1A R1673P photoreceptors but not R1664Q.

A) ERGs of 30-day-old cac mutant clones in photoreceptors and cac mutants carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) or mutant transgenes (GR-R1673P, GR-R1664Q). ***p<0.001; **p<0.01; *p<0.05; NS, not significant. p-values were consistent with those on one way ANOVA. B) Transmission electron microscopy showing the ultrastructure of cac mutant photoreceptor clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) with a normal pattern of 7 Drosophila photoreceptors per ommatidium. C) cac mutant photoreceptor clones with 7 Drosophila photoreceptors per ommatidium showing no severe degenerative changes. D) cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1664Q missense variant with a normal pattern of 7 Drosophila photoreceptors per ommatidium. E) cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1673P missense variant with dramatic neurodegeneration (red arrowheads) involving the photoreceptors. F) Quantification of degenerative rhabdomeres per ommatidium. ***p<0.001; NS, not significant. p-values were consistent with those on one way ANOVA.

Ultrastructure of Drosophila photoreceptors with CACNA1A alleles

To assess if the R1673P allele may also affect the ultrastructure of the photoreceptors, we performed transmission electron microscopy (TEM). 30-day-old cac mutant flies show a slight change in retinal morphology, but the photoreceptor cell bodies retain all seven rhabdomeres and normal overall structure compared to cac rescued by the wild type cac containing P[acman] clone (). Moreover, the cac mutants rescued with GR-R1664Q (Patients 2–5) did not exhibit obvious morphological defects of photoreceptors at 30 days (). In contrast, the cac mutant photoreceptors rescued by GR-R1673P (Patient 1) show obvious features of photoreceptor neurodegeneration (). The rhabdomeres of the photoreceptors are severely disrupted (arrows), and the cytoplasms of these cells are filled with autophagic vesicles implying neurodegeneration. The severe neurodegenerative phenotype observed here has never been seen in any cac alleles, including the null alleles, thus suggesting that R1673P may act via a (toxic) gain-of-function mechanism.

We also performed TEM at the level of the lamina, where the presynaptic photoreceptors make synaptic connections with post-synaptic neurons. These data show even more dramatic differences in phenotype between the cac mutants rescued by wild type and mutant P[acman] transgenes (). cac rescued by the wild type cac P[acman] clone displayed normal morphology of six photoreceptor terminals (green areas in ). Consistent with our previous findings [6], the cac mutant exhibited aberrantly expanded terminals, accumulation of autophagic vacuoles (AVs) and some signs of synaptic degeneration (red area in ). The P[acman] clone containing the R1664Q (Patients 2–5) variant partially rescued this terminal expansion phenotype (), whereas the cac mutant rescued by GR-R1673P (Patient 1) shows smaller size of their terminals (, Quantification in ), a phenotype which is likely due to the depletion of more synaptic vesicles with gain of channel function. In addition, we saw evidence of a dramatic synaptic degeneration in the GR-R1673P rescued flies (red outlined areas in ) compared to the other genotypes. These data indicate that the R1673P mutation causes severe neurodegeneration in both photoreceptor cell bodies and terminals, likely due to gain of channel function that is toxic to neurons during aging.

Ultrastructural neurodegeneration in CACNA1A R1673P photoreceptors but not R1664Q in the lamina layer.

A) Transmission electron microscopy showing the ultrastructure of cac mutant photoreceptor at the level of the lamina where the photoreceptor neurons synapse onto the laminar neurons. Clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) have a normal pattern of six Drosophila photoreceptors. B) cac mutant photoreceptor clones with expansion and degenerative changes. C) cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1664Q missense variant with reduced expansion. D) cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1673P missense variant with dramatic neurodegeneration evidenced by loss of the normal ultrastructure, and apparent filling with disorganized electron dense material (red outlined areas) involving the synapsing photoreceptors. E) Quantification of capitate projections (CPs), active zones (AZs), mean terminal areas, autophagic vesicles per expanded terminal, and degenerative terminals per cartridge, respectively. Data are presented as means ± SEM. p values were calculated using Student’s t test. ***p < 0.001; *p<0.05; NS, not significant. p-values were consistent with those on one way ANOVA for all groups.

Interestingly, we did not observe any severe neurodegeneration in cac (nonsense) mutants rescued by GR-R1673P in both retinae and laminae ( and ). Instead, we observed partial rescue of the terminal expansion phenotype, and notably autophagic vacuoles did not accumulate (). The latter suggests that lysosome-autophagosome fusion function is still present in cac with the R1673P mutation. The differences in the phenotypes observed in cac and cac mutants rescued by GR-R1673P further support that R1673P is a gain-of-function variant. In the cac partial loss-of-function background, the R1673P variant can generate toxic gain-of-function effects that lead to severe neurodegeneration. In contrast, the cac null allele expresses no endogenous cac and therefore alleviates the toxicity that arises from the R1673P variant. In summary, our experiments in Drosophila strongly suggest that the R1673P and R1664Q mutations are likely to be functional in human and likely to exert their effects through distinct mechanisms.

Discussion

We report five individuals with similar clinical presentations of ataxia, expressive speech delay, motor incoordination, and age of onset. The severe early-onset ataxias seen in these patients are similar to reports of severe early-onset ataxia associated with CACNA1A missense variants observed in the S4 transmembrane segment of domain III (e.g. p.I1342T, p.V1396M, and p.R1352Q) [27] or domain IV (R1664Q) [34]. Given these unique clinical features, it had been proposed that these represented loss-of-function mutations of the calcium channel [34]. These severe ataxia phenotypes were thought to represent the most severe end of the spectrum of EA2 rather than the gain-of-function mechanisms seen in hemiplegic migraines [29, 34]. However, in our series, Patient 1, a girl with a de novo R1673P variant, also exhibited a progressive cerebellar neurodegenerative process of the most severe end of the CACNA1A clinical spectrum.

Our Drosophila studies indicate distinct functional consequences when comparing R1673P and R1664Q alleles. Initially we observed the R1673P allele failed to rescue lethality whereas R1664Q partially rescued, suggesting R1673P is a more severe allele. Importantly, the R1673P allele causes a neurodegenerative phenotype based on functional and morphological criteria which was not seen in either R1664Q or in loss-of-function alleles of cac. Despite the overall clinical similarity between the patients, the two alleles exhibit dramatic functional differences in Drosophila, a functional spectrum not observed previously in severe CACNA1A variants. In retrospect, we note that Patient 1, the only subject with the R1673P variant, had progressive neurodegeneration of the cerebellum, which was a distinguishing feature between her and the other four subjects. The molecular mechanism for the special arginine at position 1673 in regulating the Ca2+ channel function remains unclear. Interestingly, the recently solved crystal structure of CaV1.1 suggests that both R1664 and R1673 are positively charged residues within the voltage sensor[43]. Since many disease-associated variants are found in positively charged residues in the S4 segment of domain IV of CACNA1A (), to explore their functional differences will be an exciting future topic to eventually establish a mechanistic model for these key residues in Ca2+ channel function.

Responsiveness to treatment and medication differs between reported cases of loss-of-function and gain-of-function CACNA1A alleles [11, 18]. Patients with EA2 and loss-of-function alleles are often responsive to acetazolamide, while patients with gain-of-function alleles and FHM may respond but generally tend to be less responsive. One would predict that calcium channel blockers might be more effective for gain-of-function alleles. Whether patients with the more severe ataxias also differ clinically in their response to treatment remains to be tested. We note that, while Patient 5 had a strong positive response to acetazolamide, Patient 1 did not respond, consistent with this observation. Indeed as a result of our study Patient 1 was started on a calcium channel blocker. In conclusion, deciphering the functional impact of these severe CACNA1A alleles may provide insight into the pathogenic mechanisms and help direct therapeutic interventions.

Materials and methods

Human studies and whole exome sequencing

All human subjects research was approved by the Institutional Review Board at Baylor College of Medicine (Studies- Undiagnosed Diseases Network protocol, 15-HG-0130, approved by the National Human Genome Research Institute IRB, and in the Baylor-Hopkins Center for Mendelian Genomics protocol, H-29697, and "Evaluation of Sequence Variants" H-22769 approved by the IRB for Baylor College of Medicine). All families gave written informed consent for whole exome sequencing and publication. All sequencing studies were performed on genomic DNA from blood samples. Patient 1 obtained trio-based exome sequencing through BHCMG [31]. In brief, for patient 1 DNA samples were obtained and prepared into Illumina paired-end libraries and whole-exome capture with BCM-HGSC core design (52 Mb, Roche NimbleGen), and then sequencing on the Illumina HiSeq 2000 platform (Illumina). The produced data were aligned and mapped to the human reference genome (hg19) through the Mercury pipeline [44]. Single-nucleotide variants (SNVs) were called with the ATLAS (an integrative variant analysis pipeline optimized for variant discovery) variant calling method and annotated by the in-house Cassandra annotation pipeline that adapts ANNOVAR (Annotation of Genetic Variants) and additional in-house tools [45-47]. De novo variants were calculated by an in-house developed pipeline (DNM-Finder) [31] for in silico subtraction of parental variants from the proband’s variants in vcf files while accounting for the read number information extracted from BAM files. Bioinformatic tools predicted conservation and pathogenicity of candidate variants, and variants were compared against both an internal database and public databases such as the Exome Aggregation Consortium (ExAC) database.

Patients 2–5 had clinical proband exome sequencing at BGL [32, 33]

Fly genetics

cac and cac mutants were isolated from a chemical mutagenesis screen as described previously [6, 48]. The mapping and sequencing of the mutants was performed as described [49]. The P[acman] BAC construct that contains the full length cac genomic region was selected from a large P[acman] library that we previously described [49]. A Transgenic line from this BAC (CH321-60D21) was generated previously and named Dp(1;3)DC131 [49] which we refer to as GR-WT in this paper.

Two point mutations (R1664Q or R1673P) were introduced into the CH321-60D21 BAC by recombineering using the modified DH10B strain SW102 and a galK positive/counter selection cassette [50]. The reagents for recombineering were obtained from Biological Resources Branch at National Cancer Institute (NCI) -Frederick. The microinjections to generate transgenic flies that contain wild type or mutant 77kb P[acman] clones were performed by GenetiVision, Houston, TX.

Lethality rescue experiments in Drosophila were done by crossing y w, cac FRT19A/FM7c or y w, cac FRT19A/FM7c with y w/Y; Dp(1;3)DC131(or -R1673P or -R1664Q) flies. The males that have no FM7c marker in the next generation were considered rescued flies. The numbers of rescued flies were also compared with FM7c males in the same progeny.

Generation of cac mutant eye clones in Drosophila

In order to circumvent the lethal phenotype, we generated mosaic clones in the Drosophila eyes for ERG and EM experiments. Virgin females of y w, cac (or cac) FRT19A / FM7c; Dp(1;3)DC131-R1673P (or +) / CyO were crossed with cl(1)* FRT19A/ Y; ey-FLP males and we examined y w, cac (or cac) FRT19A / cl(1)* FRT19A; ey-FLP/ Dp(1;3)DC131-R1673P (or +) progeny for ERG and EM defects. Homozygous mutant cells were marked by w and heterozygous cells were marked by w. Homozygous wild-type cells were eliminated by the recessive cell lethal mutation (cl(1)*) to give the mutant clones a growth advantage.

Electroretinogram (ERG) recording

ERG recordings were performed as previously described [51]. Briefly, adult flies were glued to a glass slide, a recording electrode was placed on the surface of the eye while a reference probe was inserted in the thorax. A fly eye was exposed to a flash of white light for 1 second. Responses were recorded and analyzed with AXON TM-pCLAMP8 software. Data were analyzed by two-tailed unpaired Student’s t test. A p-value of <0.05 was considered statistically significant.

Transmission electron microscopy (TEM)

Laminae in adult flies were processed for TEM imaging as described [52]. Samples were processed using a Ted Pella Bio-Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25°C in 4% paraformaldehyde, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4°C for 48 hours. 1% osmium tetroxide was used for secondary fixation and subsequently dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Science, Hatfield, PA). 50 nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Science, Hatfield, PA). Specimens were stained with 1% uranyl acetate and 2.5% lead citrate and imaged using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 megapixel CCD camera.

Drosophila genetics

The genotypes of the fly strains used are as follows:

cac y w cac

cac FRT19A/ P{w+} cl(1) FRT19A; eyFLP/+

cac Dp(1;3)DC131/+

cac,

cac,

cac,

cac,

cac.

Clinical summaries for patients 1–5.

(DOCX)

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Members of the Undiagnosed Diseases Network.

The members of the UDN listed.

(XLSX)

Click here for additional data file.

Imaging and molecular characteristics of the patients with CACNA1A de novo variants.

A) Patient 4 brain MRI at 8 years showing a mild atrophy of the cerebellar vermis. B) Patient 4 brain MRI axial image showing normal cerebellar hemispheres. C) All five subjects had de novo missense variants in CACNA1A. Sanger traces for each father, mother and proband are shown below, the red arrows indicate the position of the de novo variant.

(TIF)

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Electroretinogram recordings from the cac alleles rescued by the P[acman] constructs.

A) ERGs of 3-day-old cac mutant clones in photoreceptors and cac mutants carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) or mutant transgenes (GR-R1673P, GR-R1664Q). Similar results to those were seen in the cac background in B) ERGs of 30-day-old cac mutant clones in photoreceptors and cac mutants carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) or mutant transgenes (GR-R1673P, GR-R1664Q). ***p<0.001; *p<0.05; NS, not significant. *, p < 0.05, one-way ANOVA.

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TEM of cac photoreceptors in retinae shows no severe neurodegeneration in CACNA1A R1673P.

A. Transmission electron microscopy showing the ultrastructure of cac mutant photoreceptor clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) with a normal pattern of 7 Drosophila photoreceptors per ommatidium. B. cac mutant photoreceptor clones with 7 Drosophila photoreceptors per ommatidium showing no severe degenerative changes. C. cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1664Q missense variant with a normal pattern of 7 Drosophila photoreceptors per ommatidium. D. cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1673P missense variant with mild neurodegeneration involving the photoreceptors.

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TEM of cac photoreceptor cartridge.

A. Transmission electron microscopy showing the ultrastructure of cac mutant photoreceptor at the level of the lamina where the photoreceptor neurons synapse onto the laminar neurons. Clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) have a normal pattern of six Drosophila photoreceptors. B. cac mutant photoreceptor clones with severe expansion and accumulation of autophagic vesicles. C. cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1664Q missense variant show reduced expansion. D. cac mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1673P missense variant partially rescued cac phenotypes. E. Quantification of capitate projections (CPs), active zones (AZs), mean terminal areas, autophagic vesicles per expanded terminal, and degenerative terminals per cartridge, respectively. Data are presented as means ± SEM. p values were calculated using Student’s t test. ***p < 0.001; **p<0.01; *p<0.05; NS, not significant. ***, p < 0.001, one-way ANOVA for all groups.

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Table 1

Clinical features of the 5 individuals with de novo CACNA1A variants.

Identifier	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5
Coding variant(NM_001127221.1)	c.5018G>C	c.4991G>A
Protein variant	p.R1673P	p.R1664Q
Inheritance	De novo	De novo	De novo	De novo	De novo
Parental ages	38(mat), 38(pat)	17(mat), 20(pat)	30(mat), 33(pat)	18(mat), 22(pat)	38(mat), 41(pat)
Sex	Female	Female	Male	Male	Female
Age	8 years	5 years	5 years	8 years	6 years
Ancestry	Hispanic & European	Hispanic	European	Hispanic	European, Ashkenazi Jewish
Ataxia	Present; uses walker	Severe—independent ambulation difficult	Wide-based gait, frequent falls, significant toe walking, wears AFOs, uses adaptive stroller	Unsteady gait	Poor coordination, truncal ataxia
Cerebellar findings	Progressive cerebellar atrophy	None	None	Mild atrophy of cerebellar vermis, hemispheres normal	None
Other MRI findings	Mild thinning of the body and splenium of the corpus callosum; mild delay in deep white matter myelination	Normal MRI	Thin corpus callosum posteriorly	None	Normal MRI at 24m and 46m
Development	Global delays; no language (uses signs & iPad)	Global delays, has very few words	Global delays; walked at 27m, speech at 15m; dysarthric speech	Global delays; uses 6-7-word sentences; dysarthric; below normal IQ	Global delays (Expressive language delay, gross/fine motor delay); dysarthria, dysgrammatic speech
Hypotonia	Congenital, generalized	Some	Present	Present	Mild
Additional neurologic findings		Worsening behavioral issues; hyperreflexia	Difficulty sleeping, sensory processing disorder	Aggression	ADD/executive dysfunction; hyporeflexic
Ophthalmologic findings	Accommodative esotropia, hyperopia	Eye movement disorder	Strabismus, myopia, astigmatism	Ocular apraxia	Alternating strabismus, esotropia
Weight (percentile)	19th	90th	90th	89th
Height (percentile)	25th	70th	82nd	51st
Head circumference (percentile)	76th	75th	65th	78th
Dysmorphisms	A tented upper lip and prominent jaw	None	Mild midface hypoplasia, occipital flattening, corrected plagiocephaly		None
Additional features		Constipation	Gastroesophageal reflux disease	Hyperextensible, inverted nipples	Low CSF HIAA, hyperextensible

ADD, attention deficit disorder; CSF, cerebrospinal fluid; HIAA, Hydroxyindoleacetic acid; m, months; mat, maternal age; pat, paternal age