Introduction

Spinocerebellar ataxia type 29 (SCA29) is caused by missense variants in ITPR1 [1]. However, ITPR1 contains large numbers of missense variants of unknown significance (VUS), demonstrating the need to identify variants with high functional impact amongst the multitude of ITPR1 VUS before making a diagnosis of SCA29.

Demonstration of de novo occurrence is a powerful tool to strongly support pathogenicity of missense variants against a background of manifold rare VUS [2]. In fact, de novo variants in ITPR1 might represent a particularly frequent cause of ataxia, especially in early-onset ataxia (EOA) [3]. Understanding the variant characteristics and mechanism of action of disease-associated de novo variants in ITPR1 might guide interpretation of the functional impact of ITPR1 variants and inform the genetic diagnosis of SCA29. So far, however, neither the exact frequency nor specificity of ITPR1 de novo variants have been thoroughly determined in larger ataxia and control populations, and their functional mechanism of action in SCA29 remains unclear.

Here we aimed to systematically determine the frequency, phenotype, genetic characteristics, and functional mechanism of de novo ITPR1 missense variants in EOA in two independent cohorts from different continents. Moreover, we also aimed to determine the overall mutational burden of ITPR1 variants in EOA, including inherited ITPR1 variants. We hypothesized that (i) de novo ITPR1 missense variants are a recurrent and specific cause of EOA across independent cohorts; (ii) inherited ITPR1 missense variants might also be frequent, yet with absent or small functional or phenotypic impact; (iii) functional characterization of ITPR1 de novo variants might help to identify those ITPR1 variants with large effect size, thus facilitating the diagnosis of SCA29.

Materials and methods

Patients

Three cohorts of patients were aggregated. Cohort #1: Target cohort. N = 120 consecutive index subjects with unexplained EOA (age of onset <30 years), “sporadic” family history (i.e., no ataxia in previous or same generation), and negative for Friedreich’s ataxia repeat expansions, were recruited at the University Hospital, Tübingen, Germany, from 2010–2016. All patients originated from European, Mediterranean or Middle Eastern countries. Cohort #2: Validation cohort. This cohort was used to confirm the frequency of ITPR1 de novo variants observed in the target cohort in an independent mixed-population cohort resident in North America. N = 72 index subjects with unexplained EOA and “sporadic” family history (same criteria as above), and availability of parental DNA for trio sequencing (to confirm de novo inheritance), were compiled from consecutive referrals to Ambry Genetics, Aliso Viejo, USA, from 2012–2016. Cohort #3: Disease control cohort. This cohort served to rule out that the occurrence of ITPR1 de novo variants—observed in the target and the validation cohort—was a nonspecific finding, unrelated to ataxia. N = 139 index subjects with unexplained early-onset epileptic encephalopathies (EOEE; age of onset < 3 years) were recruited via the EuroEPINOMICS-RES network. Only subjects with “sporadic” family history (criteria see above), and availability of parental DNA for trio sequencing (to confirm de novo occurrence) were included.

Genetic screening by WES and high-throughput panel sequencing

Sequencing. Subjects from cohort#1 were screened for ITPR1 mutations by WES and/or large next-generation sequencing panels (>120 ataxia related genes); subjects from cohort #2 and cohort #3 were screened by trio WES (for methodological details, see Supplement 1). Filtering. Variants from the target cohort (cohort #1) were filtered for (i) non-synonymous heterozygous variants in ITPR1, with (ii) absence or extremely low frequency (minor allele frequency < 0.02%) in the public databases EVS6500, 1000Genomes project, and ExAC as well as in GENESIS (<11 heterozygous or homozygous alleles in 5996 subjects in GENESIS [4], (iii) at least moderate conservation (GERP score > 2 OR PhastCons score > 0.5), and (iv) at least moderate genotype quality (GQ [GATK] quality index > 35; read depth > 8X). All ITPR1 variants identified in the index patients from cohort #1 were subsequently tested in available family members by Sanger sequencing, revealing whether they occurred de novo or were parentally inherited. Variants from the EOA validation cohort (cohort #2) and the EOEE disease control cohort (cohort #3) were filtered only for ITPR1 de novo variants, as these two WES trio cohorts only served to confirm and control the frequency of ITPR1 de novo variants identified in the target cohort. Identified ITPR1 variants and associated phenotypes were submitted to the public archive ClinVar (URL:https://www.ncbi.nlm.nih.gov/clinvar/; accession numbers SCV000700178 - SCV000700190).

Exploratory mutational burden analysis

To further explore the significance of ITPR1 missense variants in EOA, we performed an exploratory analysis to determine whether ITPR1 missense variants are enriched in EOA compared to the general population. The frequency of rare, conserved ITPR1 missense variants in the EOA target cohort was compared to their frequency in ~60,606 control exomes (=121,612 alleles) from the Exome Aggregation Consortium (ExAC [5], accessed 07/2016). Identical filter settings were used to filter both datasets.

In silico characteristics of de novo vs. general ITPR1 missense variants

We next analyzed whether bioinformatic in silico characteristics of de novo ITPR1 variants differed from the in silico characteristics of inherited ITPR1 variants and of ITPR1 variants in the general population. To this end, all de novo ITPR1 variants identified in this study (cohort #1 and cohort #2) or reported previously in the literature were aggregated. They were then compared to the inherited ITPR1 missense variants (identified from cohort #1) and ITPR1 missense variants from general population databases ExAc, EVS6500, and GENESIS (see Supplement 4) for common genetic in silico characteristics.

Impact of ITPR1 missense variants on IP3-induced Ca2+ release

ITPR1 encodes an IP3-receptor acting as a Ca2+ release channel, localized predominantly in membranes of endoplasmic reticulum (ER) Ca2+ stores [6, 7]. Based on this well-established role of ITPR1, the functional effect of ITPR1 variants was investigated by assessing their impact on IP3-induced Ca2+-release in stable, inducible HEK293 cell lines that were constructed to express either one of these ITPR1 variants or wild-type. IP3-induced Ca2+ release was determined by loading the HEK293 cells with an ER luminal Ca2+ dye (mag-fluo-4 AM), and measuring Ca2+ release upon increasing concentrations of IP3 (10–1000 nM) which were consecutively added to the cells (for methodological details, see Supplement 2). We established HEK293 cell lines for 4 exemplary paradigmatic ITPR1 variants: one de novo variant from the target cohort (c.1702A>G (p.(R568G))), one de novo variant from the validation cohort (c.800C>T (p.(T267M))), one de novo variant present in both cohorts (c.805C>T (p.(R269W))), and one inherited variant with high in silico prediction scores (c.1606C>T (p.(L536F))).

Results

Frequency and specificity of de novo ITPR1 missense variants

We identified 10 ITPR1 missense variants in a total of 8 of 120 index patients from the EOA target cohort (cohort #1), yielding an ITPR1 missense carrier frequency of 6.6% (8/120) (Table 1). One patient (P1) carried 3 different ITPR1 variants (Fig. 1a). While 7/10 ITPR1 variants (70%) were parentally inherited, the remaining 3/10 variants (30%) were de novo, yielding a de novo carrier frequency of 2.5% (3/120) in our EOA target cohort. Out of the 72 index patients from the EOA validation cohort (cohort #2), four subjects carried a de novo ITPR1 variant (4/72 = 5.5%) (Table 1), thus confirming the carrier frequency observed in the target cohort. No de novo ITPR1 variant was found in the 139 patients from the EOEE disease control cohort (cohort #3) (0/139 = 0%). This demonstrates that the frequency of ITPR1 de novo variants observed in the two EOA cohorts cannot be attributed to a general high baseline prevalence of ITPR1 de novo variants, unrelated to ataxia (p = 0.046, Fisher’s exact test, 2-sided).

Table 1 Genetic characteristics of the ITPR1 missense variants identified in this study and published autosomal-dominant families
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Fig. 1
figure 1

Pedigrees of families with de novo or inherited ITPR1 missense variants from the target cohort, and cerebral MRI of ITPR1 de novo patients. A Pedigrees. Patient P1 carried 3 ITPR1 variants, two of which were inherited paternally and arranged in cis, while the third variant (c.6205G>T) was de novo. Phasing between the de novo variant and the two paternally inherited variants cannot be determined from the available data. Three families (P9/11/12) carried ITPR1 variants inherited by one parent in addition to biallelic variants in another ataxia gene (GAN, SYNE1, ATM) that fully explained the phenotype. Variants for which the phase cannot be determined are separated by a dash; dotted lines separate variants located on different chromosomes. B Sagittal (i, iii, v) and parasagittal (ii, iv, vi) T1 cerebral magnetic resonance imaging (MRI) shows mild atrophy of the anterior cerebellar cortex with enlarged spaces between foliae (particular pronounced in patient P1, ii), and of the cerebellar vermis in patients P1 (i, ii) and P2 (iii, iv), but no cerebellar atrophy in patient P7 (v, vi)

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Phenotype of patients with ITPR1 de novo variants

All 7 EOA patients with ITPR1 de novo variants (3 from cohort #1; 4 from cohort #2) presented with infantile onset cerebellar ataxia starting before the age of 2 years, including delayed motor milestones (Table 2). Cognitive deficits of variable degree were observed in 3 out of 4 patients where this information was available, reaching from only mild dyscalculia (P2) to severe intellectual disability with a speech vocabulary of only a few words (P7 at age 12 years). In contrast, patient P1 showed normal intelligence with an IQ of 97. Aniridia was noted only in 1/7 patients (P4), demonstrating that the “Gillespie syndrome” [7, 8] is a relatively infrequent presentation of ITPR1 variants. In contrast, 4/7 patients showed strabismus (Table 2), suggesting that it is a frequent feature of de novo ITPR1 ataxia. One patient (P2) also revealed congenital horizontal oculomotor apraxia (COMA [9]), i.e., slowed initiation and performance of horizontal saccades. The oculomotor apraxia slowly ameliorated throughout the following disease course, but still precludes her from obtaining driving license at her current age of 18 years. P2 also showed limb action myoclonus throughout all prospective longitudinal assessments, thus extending the movement disorder spectrum associated with ITPR1 ataxia. In contrast, none of the patients identified here showed evidence for other non-ataxia signs that are frequently observed in other EOAs [10, 11] such as epilepsy, pyramidal tract affection or peripheral neuropathy (Table 2). Cerebellar atrophy was seen only in 2 out of 5 patients where MRI was available (Fig. 1b, Table 2), suggesting that it might not be an obligate feature of de novo ITPR1 ataxia, at least not early in the disease course. One patient (P6) showed no cerebellar, but cerebral atrophy, suggesting that not only cerebellar, but also cerebral cortical neurons are susceptible to ITPR1 dysfunction (for further discussion on the MRI findings and the clinically non-progressive disease course, see Supplement 3).

Table 2 Clinical, imaging, and electrophysiological features of de novo ITPR1 ataxia
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An enrichment of ITPR1 variants in EOA

Our exploratory mutational burden analysis showed an enrichment of ITPR1 missense variants in the EOA target cohort compared to the general population (odds ratio 4.27, p = 0.0002, 95% confidence interval 2.013–8.014), which prevailed even after removing the ITPR1 de novo variants (odds ratio 2.95, p = 0.01181, 95% confidence interval 1.170–6.188). At least some of the inherited ITPR1 variants might therefore possibly contribute to the EOA phenotype, acting e.g., as risk factors or low-penetrance alleles (for a detailed presentation of the results and further discussion see Supplement 5).

Characteristics of inherited ITPR1 missense variants and of ITPR1 missense variants in the general population

We next explored the 7 inherited ITPR1 variants from cohort #1 in more detail. In line with the inclusion criteria of only sporadic index patients, all parents of the patients with inherited ITPR1 variants were healthy, without even mild cerebellar signs, demonstrating lack of a clinical phenotype for these 7 inherited variants in the parental generation. 3/6 patients with an inherited ITPR1 variant carried damaging biallelic variants in other well-established EOA genes (SYNE1, GAN1, ATM; Table 1 and Fig. 1a) which fully explained the phenotypic presentation. The subject carrying two inherited ITPR1 variants in addition to one de novo variant (patient P1) did not show a more severe disease phenotype or progression than the subjects carrying only one de novo variant. Rather, her phenotype was similarly mild as the phenotype seen in patient P2 and she even improved over time. Compared to the severly affected patient P7 her phenotype was much less severe, indicating that the two inherited ITPR1 variants additionally identified in P1 have no major effect on the phenotype. Taken together, these findings suggest that the majority of the inherited ITPR1 missense variants likely have minimal or no impact on the clinical phenotype.

Screening of >70,000 WES from the three general population databases identified 550 rare, well conserved ITPR1 missense variants (see Supplement 4), supporting the notion that many of these variants might indeed be associated with a minimal or no direct effect on a particular clinical phenotype. Common bioinformatics tools did not allow to distinguish the inherited ITPR1 variants from cohort #1 and the ITPR1 variants observed in the general population (many of them likely with limited or no direct effect) from ITPR1 de novo variants (most of them likely with a strong effect). All de novo variants, but also many of these other variants were: (i) very rare or even private according to ExAC, EVS, and Genesis; (ii) highly conserved according to PhastCons or GERP scores (Fig. 2c); (iii) predicted to be “deleterious” by commonly used in silico prediction tools like PolyPhen2, PROVEAN, SIFT [12] and CADD scores (Combined Annotation Dependent Depletion) [13] (Fig. 2b); and (iv) located in functional domains like e.g., the MIR, RIH or Ion-TM domains (Fig. 2a). This suggests that while bioinformatics tools might be helpful to discard those ITPR1 missense variants which likely have no effect (namely variants which are frequent, not conserved, and predicted to be tolerated), they do not suffice in themselves to demonstrate whether a certain variant has a sufficiently deleterious effect to cause a phenotype or not.

Fig. 2
figure 2

A Schematic representation of the ITPR1 structure and location of ITPR1 variants. ITPR1 transcript variant NM_001099952/ENST00000423119 encodes a protein of 2710 amino acid residues length (Q14643-3). The ITPR1 protein contains several conserved protein domains (NCBI conserved domains database): an inositol 1,4,5-trisphosphate/ryanodine receptor domain (Ins145, aa4-225) for inositol 1,4,5-trisphosphate binding, a MIR domain (aa 232-433), that conveys ligand transferase function, two RIH domains (aa 472-677, aa1176-1355), a ryanodine receptor homology domain (RIH_assoc, aa1923-2035) and a transmembrane ion transport domain (Ion-TM, aa2235-2550) containing six putative transmembrane motifs. Both de novo (red circles and stars) and autosomal dominant (AD, yellow circles and stars) variants associated with ataxia are spread over most part of the protein; the variant associated with the unique phenotype (infantile onset non-progressive ataxia, pontocerebellar hypoplasia) described by van Dijk et al. [21] is marked by a blue border. Missense variants associated with infantile onset ataxia without iris hypoplasia (marked by circles) tend to cluster in the N-terminal part of the protein, preferentially the Ins145 domain, the MIR domain, and the N-terminal RIH-domain. In contrast, variants leading to ataxia complicated by iris hypoplasia (Gillespie syndrome, marked by stars) exclusively locate to the C-terminus. However, as also shown here, not all variants at this C-terminal end include iris hypoplasia (see red circle in Ion-TM domain, indicating patient P7). All ITPR1 variants reported to cause autosomal recessive Gillespie syndrome are truncating in nature (orange stars or circles). All but one variant are associated with infantile onset ataxia; the autosomal-dominant variant p. (P1074L) that reportedly causes adult onset ataxia is circled by a gray border. Rare conserved variants reported in public databases (grey dots; EVS, ExAC, Genesis) are equally spread over the whole protein and don’t appear to spare certain functional domains.  B, C Rare conserved variants were extracted from public databases (EVS, ExAC, Genesis) and annotated with four established scores predicting the deleteriousness of variants (PROVEAN, SIFT, PolyPhen-2, CADD) B and two scores evaluating the conservation (GERP, PhastCons). The ‘deleterious’ range of each in silico prediction score is marked by an orange shaded region (cutoff PROVEAN < −2.5, SIFT < 0.05, PolyPhen-2 > 0.85, CADD > 15). Although most de novo (red circles) and AD (yellow circles) variants cluster in the ‘deleterious’ range for each score, none of the scores discriminates well between ataxia-associated variants and variants retrieved from public databases (gray dots). C The same holds true for the conservation scores (GERP, PhastCons). Although most de novo (red circles) and AD (yellow circles) variants cluster in the highly conserved range for each score, neither one of the scores per se nor the combination thereof discriminates well between ataxia-associated variants and variants retrieved from public databases (gray dots). Please note that no variants were shown in the low range of the conservation scores (lower left quarter) as we only filtered for variants with a score PhastCons > 0.5 OR GERP > 2. D De novo and AD variants associated with ataxia tend to cluster at the extreme end of both the SIFT and PolyPhen-2 score. We therefore evaluated whether a combination of both scores can be used to predict deleteriousness of ITPR1 variants. While a ‘tolerated’ prediction in SIFT and polyphen-2 are a strong indicator against pathogenicity of an ITPR1 missense variant, ‘deleterious’ predictions in these two scores do not reliably discriminate de novo ITPR1 variants from ITPR1 missense variants in public databases

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Functional effect of ITPR1 missense variants on IP3-induced Ca2+release

We investigated the functional effect of selected ITPR1 variants from both the EOA target cohort (cohort#1) and EOA validation cohort (cohort#2) on IP3-induced Ca2+ release in HEK293 cells. Compared to wildtype, all three HEK293 cell lines expressing a de novo ITPR1 variant (c.800C>T (p.(T267M)); c.805C>T; (p.(R269W)) or c.1702A>G (p.(R568G))) showed a markedly reduced fractional Ca2+ release (i.e. smaller drop in the steady-state ER Ca2+ level) upon induction by IP3 (Fig. 3a, b) (p < 0.001). This demonstrates a (strong) loss-of-function effect as the mechanism of action of these de novo ITPR1 variants. In contrast, the IP3 response of the HEK293 line expressing the inherited ITPR1 variant (c.1606C>T (p.(L536F))) did not differ from wildype (p = n.s.), yet differed significantly from the 3 de novo ITPR1 variants (p < 0.001) (Fig. 3a, b). Thus, functional analyses can help to disentangle those ITPR1 missense variants with a large effect size from those ITPR1 variants with no or only minimal effect on Ca2+ release.

Fig. 3
figure 3

IP3-induced Ca2+ release in HEK293 cells expressing ITPR1 wildtype versus ITPR1 variants. A Stable, inducible HEK293 cells expressing the ITPR1 wildtype (WT) or ITPR1 variants were loaded with an ER luminal Ca2+ dye (mag-fluo-4). Fluorescence intensity of mag-fluo-4 loaded cells expressing ITPR1-WT (black trace), c.800C>T (p.(T267M)) (red); c.805C>T (p.(R269W)) (pink), c.1606C>T (p.(L536F)) (orange); or c.1702A>G (p. (R568G)) (green) was measured continuously before and after repeated additions of various concentrations of IP3 (10, 30, 100, 300, and 1000 nM), followed by addition of 1000 nM IP3 plus 10 µM tBHQ. The steady-state mag fluo-4 signal before the first addition of IP3 was taken as the maximum ER Ca2+ level (Fmax, 100%), whereas, the steady-state mag fluo-4 signal after the addition of IP3 (1000 nM) plus tBHQ (10 µM) was taken as the minimum ER Ca2+ level (Fmin, 0%). Compared to WT, all three HEK293 cell lines expressing a de novo ITPR1 variant (c.800C>T (p.(T267M)); c.805C>T; (p.(R269W)); or c.1702A>G (p.(R568G))) showed a reduced fractional Ca2+ release (i.e., smaller drop in the steady-state ER Ca2+ level) upon induction by IP3. In contrast, the IP3 response of the HEK293 cells expressing the inherited ITPR1 variant (c.1606C>T (p.(L536F))) did not differ from wildype (p = n.s.), yet differed significantly from the three de novo ITPR1 variants (p < 0.001).  B The steady-state ER Ca2+ levels (%) at different cumulative concentrations of IP3 in HEK293 cells expressing ITPR1 WT or variants were determined and normalized to the maximum ER Ca2+ content (Fmax–Fmin). Data shown are mean ± SEM (n = 4–6) (*P < 0.001 vs WT or c.1606C>T (p.(L536F)))

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Discussion

De novo variants in ITPR1/SCA29 are a recurrent cause of EOA, not observed in other early-onset neurological disease

Here we report the first systematic and largest screening series of ITPR1 patients in several independent cohorts. We show that de novo missense variants in ITPR1/SCA29 account for a substantial share of patients with so far unexplained sporadic ataxia, yielding an estimated frequency of 2.5% to 5.5% of sporadic EOA in Europe and North America. The high number of de novo variants identified in our EOA cohorts (seven de novo mutations, four of them reported here the first time) demonstrates that not only autosomal-recessive variants, but also de novo variants in SCA genes need to be appreciated as a recurrent cause of EOA. Our finding receives particular support from its validation in a second, independent large screening cohort, and from the use of a specific neurologic control cohort (EOEE) where a high share of de novo variants can generally be expected [14]. Exploiting this disease control cohort, we show that the increased frequency of ITPR1 de novo variants is not due to a ubiquitous high frequency of de novo variants in ITPR1 in neurologic populations, unrelated to ataxia. At the same time, this finding shows that—unlike many other de novo variants in ataxia genes, which might also present with prominent epileptic encephalopathy phenotypes (e.g., KCNA2 [15, 16] or KCND3 [17])—de novo variants in ITPR1 do not seem to present a common cause of epileptic encephalopathy. This is also of particular interest as epileptic seizures are a major phenotypic feature in the mouse model of ITPR1 [18].

Loss of channel function: a common mechanism of ITPR1 de novo missense variants causing SCA29

The mechanism of action of ITPR1 variants causing SCA29 has not yet been systematically studied in a larger series. Our findings demonstrate that loss of the IP3 channel function, demonstrated by impairment of the IP3-induced endoplasmic reticulum Ca2+ release, is a common mechanism associated with ITPR1 variants causing SCA29, here observed in all 3 of 3 de novo variants that were analyzed, including the recurrent R269W variant which we and others [19, 20] have identified in different cohorts. This loss of IP3 channel function could very well be a result of a dominant-negative mode of action of mutant IP3 subunits on the tetrameric IP3 channel, as speculated by McEntagart et al. [8]. This does, of course, not exclude the possibility that also gain of IP3 channel function might lead to SCA29 in some cases with other ITPR1 variants.

Our findings might help to guide future pharmacological therapies for SCA29, prioritizing those compounds that enhance the sensitivity of endoplasmic reticulum Ca2+ release and thus compensate for the loss of channel function effect of SCA29-associated ITPR1 variants.

Extending and specifying the phenotype of de novo ITPR1 ataxia

Our phenotypic findings show that the onset of de novo ITPR1 ataxia is limited to the infantile end of the EOA disease spectrum (i.e., it does not start at >2 years of age) and is frequently complicated by cognitive deficits. This phenotypic combination makes it less likely that affected subjects will reproduce, which might explain why the majority of the published cases with ITPR1-associated ataxia are de novo, while descriptions of autosomal dominant pedigrees are relatively sparse.

Moreover, our phenotypic results show that the oculomotor presentation of ITPR1 can also encompass congenital oculomotor apraxia, elaborating on a previous report of de novo ITPR1 patients [3]. Our findings thus extend the genetic basis of the well-recognized syndrome of “congenital oculomotor apraxia” (COMA [9]; OMIM #257550), where the genetic background beyond Joubert syndrome had still remained largely elusive [9].

Phenotype-genotype relations in ITPR1 de novo ataxia

Our findings confirm the classic Gillespie cluster (=EOA plus aniridia; OMIM #206700) as a striking (albeit overall relatively infrequent; 1/7 = 14% of the cases) phenotype of ITPR1 variants (patient P4) [7, 8]. In fact, P4 carried the same variant as observed previously in four independent subjects with this particular phenotypic cluster (c.7687_7689delAAG (p. (K2563del)), referred to as NM_001168272.1:c.7786_7788delAAG, p.Lys2596del in [8]) [7, 8], thus indicating a relatively high degree of genotype–phenotype correlation of this particular variant with the classic Gillespie cluster. Variants associated with the Gillespie syndrome cluster at the C-terminus at protein positions >2000, with predilection for the transmembrane ion transport domain (see Fig. 2a). However, as also shown here, not all variants at this C-terminal end are associated with iris hypoplasia (see patient P7, Fig. 2a). In contrast to the variants associated with the Gillespie cluster, variants associated with congenital ataxia without iris hypoplasia tend to cluster in the N-terminal part of the protein (preferentially the Ins145 domain, the MIR domain, and the N-terminal RIH-domain, see Fig. 2a).

Apart from this partial genotype–phenotype association, phenotypic variability in de novo ITPR1 ataxia can be substantial. Two subjects carried an identical variant (c.805C>T; p.(R269W)), but showed very different phenotypes and disease severity (P2: mild ataxia, isolated mild cognitive deficits, mild vermian cerebellar atrophy; P6: severe ataxia, severe mental retardation; no cerebellar atrophy).

Functional confirmation is warranted to disentangle disease-causing ITPR1 missense variants

Although rare and well conserved ITPR1 missense variants are enriched in EOA, we here show that they also frequently occur in the general population (~1%). Caution therefore should be used in claiming pathogenicity of ITPR1 missense variants and, consequently, in diagnosing SCA29. It demonstrates the need to disentangle those ITPR1 missense variants with a large effect on channel function (and thus high likelihood to produce a phenotype) from those ITPR1 missense variants with no or only minimal effect-size (and thus likely without direct contribution to the phenotype).

As shown here, commonly used in silico criteria (e.g., variant frequency, evolutionary conservation, domain location, or combinations thereof) alone do not suffice to distinguish between these types of ITPR1 missense variants. Correspondingly, these bioinformatic criteria did also not allow to distinguish the three (de novo) ITPR1 missense variants functionally shown to have a large effect-size from the ITPR1 missense variant (c.1606C>T (p.(L563F))) shown to have no effect. In contrast, this effect difference could be well demonstrated by a functional analysis of the IP3-induced Ca2+ release.

In conclusion, we suggest that, at least for inherited ITPR1 variants, functional confirmation of the variant effect on IP3 channel function, e.g., demonstrated by alterations of IP3-induced Ca2+-release from the ER, should be obtained before making a SCA29 diagnosis and should thus become state-of-the-art. The functional impact observed in ITPR1 de novo variants (strong loss of channel function) might thereby serve as a paradigmatic functional pattern to identify those ITPR1 variants with large effect sizes.