Characterisation of a Missense Variant of the Alström Syndrome Centrosome and Basal Body Associated Protein (ALMS1) Gene Associated with Cardiomyopathy Using Induced Pluripotent Stem Cells

TL;DR

This study uses stem cells to investigate how a specific genetic variant in the ALMS1 gene affects heart cell development and function in a model of Alström syndrome.

Contribution

The first hiPSC line based on a missense variant of ALMS1 is characterized, focusing on its impact on cardiomyocyte differentiation.

Findings

The ALMS1:c.10004G>C variant reduces cardiomyocyte differentiation efficiency in hiPSCs.

The variant does not affect contractile frequency or structural properties of formed cardiomyocytes.

The study supports avoiding the use of the ALMS1:c.7384G>C variant in cat breeding until its pathogenicity is confirmed.

Abstract

Background/Objectives: Human induced pluripotent stem cell (hiPSC) models provide a unique platform for testing the effect of genomic variants identified in patients with inherited diseases. In Alström syndrome, a rare multisystem disorder mainly caused by nonsense mutations in the ALMS1 gene, patients often present with infantile cardiomyopathy, retinal dystrophy, type 2 diabetes, and hearing loss in addition to obesity. These diverse clinical manifestations highlight the pleiotropic functions of ALMS1 in cellular processes such as ciliary signalling, cell cycle regulation, and tissue homeostasis. In cats, the ALMS1:c.7384G>C missense variant has been associated with cardiomyopathy in the absence of other symptoms of Alström syndrome, raising questions regarding the impact of this variant on cardiac pathology. Methods: To answer these questions, we generated an hiPSC line carrying the human ALMS1:c.10004G>C missense variant, homologous to the ALMS1:c.7384G>C feline variant, as well as an isogenic control, to investigate the impact of this variant on cardiomyocyte differentiation and function. Results: The introduction of the ALMS1:c.10004G>C variant in the homozygous state in hiPSCs resulted in a significant reduction in cardiomyocyte differentiation efficiency. However, the variant did not affect contractile frequency, sarcomere organisation, sarcomere length, or cardiomyocyte cell size. Together, these results suggest that while the ALMS1:c.10004G>C variant impairs cardiomyocyte differentiation, it does not disrupt the structural or functional properties of the hiPSC-derived cardiomyocytes that do form. Conclusions: We have generated and initiated the characterisation of the third ALMS1 mutant hiPSC line and the first line based on a missense variant, but further research is needed on its relevance in modelling ALMS1-related changes. Our results also support the previous recommendation not to use ALMS1:c.7384G>C for the selection of breeding cats until further data confirm its intrinsic pathogenicity.

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1. Introduction

Human induced pluripotent stem cells (hiPSCs) have proven to be a relevant model for studying hereditary disorders [1,2]. In the field of heart disease, cardiomyocytes derived from hiPSCs (hiPSC-CM) can be used, among others, to study the structural and functional consequences of genetic variants associated with isolated or syndromic cardiomyopathies in patients [3]. Among syndromic cardiomyopathies, an infantile-onset dilated cardiomyopathy is described in Alström syndrome, a rare multisystem disorder caused by mutations in the ALMS1 (ALMS1 Centrosome And Basal Body Associated Protein) gene. The syndrome is characterised by a variety of clinical manifestations, including retinal dystrophy, dilated cardiomyopathy, type 2 diabetes, hearing loss, and multi-organ fibrosis, in addition to obesity [4,5,6]. Recent discoveries have demonstrated the role of ALMS1 in the formation and function of cilia, leading to the classification of the syndrome as a ciliopathy. However, the pathophysiology and roles of ALMS1 are poorly understood, especially regarding the cardiac manifestations [7]. The diverse clinical manifestations of Alström syndrome highlight the pleiotropic functions of ALMS1 in cellular processes including ciliary signalling, cell cycle regulation, and tissue homeostasis [8,9,10,11]. Disruption of these pathways links ALMS1 not only to ciliopathy but also to fibrosis [12,13], a key pathological feature of Alström syndrome that contributes to cardiac, renal, and hepatic dysfunction [14]. In the heart, ALMS1 pathogenic variants have been strongly associated with dilated cardiomyopathy and myocardial fibrosis [15,16]. However, the precise underlying molecular mechanisms remain incompletely understood. Moreover, the genotype–phenotype correlation of ALMS1 variants has not been firmly established. While loss-of-function variants are strongly linked to the classical multisystem presentation of Alström syndrome [7,17], the impact of specific missense variants remains less clear. Some alleles may exert tissue-specific or modifier effects, influencing selected aspects of cardiac or metabolic biology without producing the full spectrum of syndromic features [18]. In this context, modelling the cardiac manifestations of Alström syndrome at the cellular level and exploring missense variants in ALMS1 appear to be two relevant approaches to filling these gaps [7]. We combined these two approaches for an ALMS1 missense variant described to be associated with an isolated cardiomyopathy in cats. A missense variant, ALMS1:c.7384G>C [ALMS1:p.(G2462R)], homologous to human ALMS1:c.10004G>C [ALMS1:p.(G3335R)], has been reported for feline hypertrophic cardiomyopathy [19,20,21]. Interestingly, affected cats do not display other systemic features typically associated with Alström syndrome.

In this study, we generated an hiPSC line carrying the ALMS1:c.10004G>C variant using CRISPR-Cas9 base editing and compared it with the isogenic control. We investigated its capacity to differentiate into cardiomyocytes, and assessed their contractile properties, sarcomere organisation, and morphology.

2. Materials and Methods

2.1. Human iPSC Generation and Culture

The human induced pluripotent stem cell (hiPSC) line AG08C5 was generated from primary foreskin fibroblasts (Coriell Institute, ref. AG08498, Camden, NJ, USA) by lentiviral transduction with a polycistronic vector encoding OCT4, KLF4, SOX2, and c-MYC (OKSM vector; Millipore, Burlington, MA, USA), as previously described by Badja et al. [22]. The AG08C5 line is registered with the French Ministry of Health (CODECOH DC-2022-5055; https://hpscreg.eu/cell-line/PGNMi001-A, accessed on 3 February 2026).

Cells were plated at 21,000 cells/cm^2^ density on a Matrigel^®^ (Corning, New York, NY, USA)-coated dish in StemMACS™ iPS-Brew XF medium (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) at 37 °C with 5% CO_2_ and 95% relative humidity. Medium was changed daily. Passaging was performed at 70–80% confluency using TrypLE™ Express (Gibco Thermo Fisher Scientific, Waltham, MA, USA) or Gentle Cell Dissociation Reagent (GCDR; STEMCELL Technologies, Vancouver, BC, Canada), depending on experimental requirements.

For cryopreservation, hiPSC culture at 70–80% confluency was washed with Phosphate Buffered Saline (PBS) and treated with 1 mL GCDR for 3–4 min at 37 °C. Cells were gently detached in StemMACS™ iPS-Brew XF medium, pelleted at 200× g for 4 min, and resuspended in StemMACS™ CryoBrew freezing medium (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) at ~1 × 10^6^ cells/cryovial. Cryovials were stored at −80 °C for 24–48 h before transferring to liquid nitrogen (−196 °C).

For thawing, cryovials were rapidly warmed at 37 °C until a small ice crystal remained. Cells were transferred to pre-warmed StemMACS™ iPS-Brew XF medium supplemented with 10 µM Y-27632 (ROCK inhibitor, Tocris, Bristol, UK) and centrifuged at 200× g for 4 min. The pellet was resuspended in fresh medium containing Y-27632 and plated on Matrigel^®^-coated dishes.

2.2. Generation of AG08C5-Cas9i iPSC Line

The AG08C5 hiPSC line was transduced with a lentiviral vector encoding a doxycycline-inducible Cas9 cassette, yielding the AG08-Cas9i line. The transduced cells were selected with treatment with puromycin (1 µg/mL) for 3 days. After amplification of the transduced cells, clonal dilution was performed, and we selected a Cas9-inducible clone by treating it with doxycycline (1 μg/mL) for 24 h, as determined by a Western blot assay using anti-Cas9 (Abcam, Cambridge, UK). The intact karyotype of this inducible line enabled its preservation for future use.

2.3. CRISPR-Cas9 Genome Editing of ALMS1

Cas9 expression was induced with 1 µg/mL doxycycline (Sigma-Aldrich, St. Louis, MO, USA) for 24 h before genome editing. The ALMS1:c.10003G>C variant (ALMS1 transcript: ENST00000613296.6, https://www.ensembl.org/index.html) was introduced into AG08-Cas9i hiPSCs using CRISPR-Cas9-mediated homology-directed repair (HDR). Following dissociation with TrypLE™ Express (Gibco, 2604013, Gibco Thermo Fisher Scientific, Waltham, MA, USA), cells were pelleted at 200× g for 4 min and resuspended in Opti-MEM^®^ Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA, USA) at 1 × 10^6^ cells/mL. For electroporation, 1 × 10^6^ cells were mixed with 5 µg of a plasmid encoding a guide RNA targeting the ALMS1:c.10003G>C locus and enhanced green fluorescent protein reporter, together with 5 µg of a single-stranded oligodeoxynucleotide HDR template (Figure S1). Electroporation was performed at 125 V for 5 ms (single pulse), using NEPA 21 (NepaGene^®^, Ichikawa, Chiba, Japan). Cells were plated into a single well of a 6-well plate and cultured for 48 h to allow recovery and GFP expression. GFP-positive cells were sorted by fluorescence-activated cell sorting using BD FACS Aria (BD Biosciences, Franklin Lakes, NJ, USA) and plated as single cells in 96-well plates and onto 100 mm dishes for colony formation. The colonies were expanded in 24 well plate later on. After clonal expansion, genomic DNA was extracted, and Sanger sequencing was performed to identify clones harbouring the targeted mutation (Table S1; Figure S1). Both cell lines tested negative for mycoplasma contamination and were cryopreserved to create a master and working cell bank. The cell lines used for experiments were between passages 12 and 22.

2.4. Human iPSC Quality Assessment

The pluripotency was assessed using flow cytometry. The cells were rinsed with 1× PBS and detached using Accutase. A single-cell suspension was made to obtain ~0.5 to 1 million cells per condition. The cells were stained with two pluripotency markers, anti-TRA-1 (Miltenyi Biotech, Bergisch Gladbach, North Rhine-Westphalia, Germany) and anti-SSEA-4 (Miltenyi Biotech, Bergisch Gladbach, North Rhine-Westphalia, Germany), with live/dead staining (ThermoFisher, Waltham, MA, USA). For each condition, single-stained and unstained cells were used as controls to set up the gating strategy.

2.5. Cardiomyocyte Differentiation

hiPSCs were plated on Matrigel^®^-coated dishes and cultured in StemMACS™ iPS-Brew XF medium. The cardiac differentiation was carried out using the Wnt modulation protocol described by Lian et al. [23]. This method predominantly yields ventricular cardiomyocytes. On day 30, cells were harvested for RNA extraction or dissociated with TrypLE™ Express for plating onto Ibidi chamber slides (Ibidi GMBH, Gräfelfing, Germany) coated with Matrigel^®^ for immunocytochemistry. The dissociated hiPSC-derived cardiomyocytes were plated at a density of 10,000 cells/cm^2^. The dissociated cardiomyocytes were cultured for 7 days in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with B27 + insulin (Thermo Fisher Scientific, Waltham, MA, USA) before fixation.

2.6. Assessment of Contraction Frequency

Spontaneous contractions were recorded using the 10× objective on an Olympus brightfield microscope. Videos of 40–60 s were acquired at a frame rate of 4 frames per second (fps). Individual contraction events were identified and counted, and contraction frequency was calculated based on the total recording duration.

2.7. Immunocytochemistry

Cells were rinsed with PBS and fixed in 4% paraformaldehyde containing 4% sucrose in PBS for 20 min at room temperature. After three PBS washes, cells were permeabilised with 0.5% Triton X-100 in PBS and blocked in 1% bovine serum albumin (BSA) for 30 min at room temperature. Samples were incubated with primary antibodies (Table S2), diluted in 1% BSA, overnight at 4 °C. After three PBS washes, secondary antibodies (Table S2) in 1% BSA were applied for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The images were acquired using a Nikon AX Confocal Microscope (Nikon, Tokyo, Japan).

Images acquired after immunostaining were used to assess differentiation efficiency. hiPSC-derived cardiomyocytes at 30 days of differentiation were immunolabeled for cardiac troponin and counterstained with DAPI. Images were acquired from 8–10 randomly selected fields per well. The total number of nuclei (DAPI-positive) and cardiac troponin-positive cells were quantified, and differentiation efficiency was calculated as the percentage of cardiac troponin-positive cells relative to the total number of nuclei.

The morphology was assessed using the Image J freehand marking tool. The area of the cardiomyocytes was marked based on the cardiac troponin skeleton, and the area of nuclei was demarcated by DAPI staining. Using the measure tool, the area was calculated.

2.8. RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR

RNA was extracted using TRIzol™ reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol, with chloroform phase separation and isopropanol precipitation. Pellets were washed twice with 70% ethanol, air-dried, and resuspended in nuclease-free water (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration and purity were determined using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesised from 500 ng total RNA using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) with random primers, following the manufacturer’s protocol. Quantitative real-time PCR (qPCR) was performed on a Bio-Rad CFX96 Real-Time PCR Detection System using SYBR^®^ Green (Roche, Basel, Switzerland). Thermal cycling conditions were as follows: 95 °C for 10 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s. This was followed by melt curve analysis from 65 °C to 95 °C in 0.5 °C increments. Normalisation was performed using GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), TBP (TATA-Box Binding Protein), and HPRT (Hypoxanthine Phosphoribosyltransferase) as housekeeping genes. Primers are shown in Table S1.

2.9. Statistical Analysis

For comparison of two groups, data were assessed for normality using the Shapiro–Wilk test. Data that were not normally distributed underwent Mann–Whitney tests, whereas data normally distributed underwent unpaired t-tests. Data were analysed and presented using GraphPad Prism version 8.4 (Dotmatics) and the tests used are mentioned in the figure legends.

3. Results

3.1. Generation of ALMS1:c.10004G>C hiPSC Lines

To explore the effect of the ALMS1:c.7384G>C [ALMS1:p.(G2462R)] feline variant associated with hypertrophic cardiomyopathy in cats and predicted in silico to be deleterious (Figure S1), we generated a human cellular model carrying the *ALMS1:*c.10004G>C variant [ALMS1:p.(G3335R)], which is homologous to the ALMS1:c.7384G>C feline variant (Figure S1), using CRISPR-Cas9 base editing in hiPSC lines. We identified a homozygous clone for ALMS1:c.10004G>C (hereinafter referred to as ALMS1-mutant, Figure S1). Both the ALMS1-mutant and control hiPSC lines were assessed for maintenance of pluripotency using SSEA-4 and TRA-1 markers, and we confirmed that genome editing and clonal selection did not compromise the pluripotent state of the hiPSCs (Figure S2).

3.2. ALMS1:c.10004G>C Variant Reduces Cardiomyocyte Differentiation Efficiency but Not Contractile Frequency

Following the establishment of the ALMS1-mutant and isogenic control hiPSC lines, we first assessed the mitotic nuclei in proliferating hiPSCs. We observed no significant difference in the proportion of mitotic nuclei between ALMS1-mutant and isogenic control hiPSCs (Figure S3). We then performed cardiomyocyte (hiPSC-CM) differentiation. The hiPSCs were first directed toward cardiac mesoderm, followed by specification into cardiomyocytes, and maintained in culture for 30 days (Figure 1a and Figure S4). Ventricular identity of most of the hiPSC-CMs has been assessed by analyzing the level of mRNA expression of the ventricular marker MYL3 (Myosin Light Chain 3). We confirmed that isogenic control and ALMS1-mutant cardiomyocytes generated under our conditions exhibited a ventricular-like molecular profile (Figure S4). Quantification at day 30 of immunolabelled cells for cardiac troponin T and DAPI (Figure 1b) revealed a significant reduction in cardiomyocyte yield in the ALMS1-mutant line compared to the isogenic control (p = 0.01; Figure 1c). We also quantified the cardiac troponin mRNA expression at different days of differentiation, and no significant differences were detected between the ALMS1-mutant line compared to the isogenic control (Figure S5).

To assess the effect of the ALMS1:c.10004G>C variant on cardiomyocyte contractility, we measured the contraction frequency in the hiPSC-CMs. The contractile activity was recorded over 100 frames, and the displacement of a defined region of interest was tracked to calculate beat frequency (Hz). A comparison between the hiPSC lines revealed no significant difference in contraction frequency (Figure 1d).

3.3. Gross Morphology of ALMS1-Mutant hiPSC-CMs Remains Unchanged

To further assess the morphological properties of the ALMS1-mutant hiPSC-CMs, we quantified the cardiomyocyte (i.e., cardiac troponin T-expressing cells) area and nucleus area (without taking into account the number of nuclei per cardiomyocyte), using ImageJ 1.38k software (Figure 2). No significant differences were observed between the ALMS1-mutant and isogenic control hiPSC-CMs for cell area (Figure 2c), but we observed a difference for nucleus area (p = 0.017; Figure 2d).

We next assessed the impact of the ALMS1:c.10004G>C variant on the nucleation state of hiPSC-CMs. Quantification revealed that the proportions of mononucleated, binucleated, and multinucleated cardiomyocytes were comparable in the ALMS1-mutant and isogenic control lines (Figure 3).

3.4. Sarcomere Organisation and Length Are Preserved in ALMS1-Mutant hiPSC-CMs

To evaluate the impact of the ALMS1:c.10004G>C variant on sarcomere formation, hiPSC-CMs at day 30 of differentiation were enzymatically dissociated and replated at low density to obtain monolayer cultures (Figure 4a). In cells immunolabelled for cardiac troponin, α-actinin, and DAPI, quantification of sarcomere organisation showed no difference in the proportion of cardiomyocytes with organised sarcomeres between the ALMS1-mutant and isogenic control hiPSC-CMs (Figure 4b,c). Further analysis of the sarcomere length also revealed no significant differences between the ALMS1-mutant and control lines (Figure 4d,e).

4. Discussion

Human induced pluripotent stem cell models provide a unique platform to test the cellular effects of genomic variants associated with diseases in both human and animal models. By introducing precise genetic variants into an otherwise identical genomic background, isogenic hiPSC lines allow the dissection of subtle phenotypic effects in a controlled setting [1,2]. Directed differentiation of hiPSCs into cardiomyocytes further enables investigation of how variants influence cardiac lineage commitment, contractility, sarcomere organisation, and cell morphology. Importantly, hiPSC-derived cardiomyocytes resemble foetal or early-stage cells [24,25,26], which may provide insight into developmental windows of vulnerability that precede obvious disease phenotypes observed later in life.

We generated human iPSC lines carrying the ALMS1:c.10004G>C variant in the homozygous state and an isogenic control to investigate the impact of the variant on cardiomyocyte differentiation and function. First, we observed no significant difference in the proportion of mitotic nuclei between ALMS1-mutant and isogenic control hiPSCs, indicating that the ALMS1:c.10004G>C variant does not affect proliferative capacity at the pluripotent stage. Differentiation of these hiPSCs into cardiomyocytes revealed a significant reduction in differentiation efficiency in the ALMS1-mutant line, indicating that the ALMS1:c.10004G>C variant may impair early cardiac lineage commitment or the survival of differentiating cardiomyocytes. Despite this effect on differentiation, the ALMS1-variant did not alter key structural or functional properties of the cardiomyocytes that were successfully generated. The contractile frequency, sarcomere organisation, sarcomere length, and cell size were all comparable between the mutant and control hiPSC-CMs. These findings suggest that once cells overcome the differentiation bottleneck, the ALMS1:c.10004G>C variant does not compromise the intrinsic functional or structural integrity of cardiomyocytes under baseline conditions. We also observed that the ALMS1:c.10004G>C variant does not alter the nucleation profile of hiPSC-CMs under the conditions examined. Most of the ALMS1-mutant and isogenic control hiPSC-CMs were mononucleated. This is consistent with published data [27] indicating that hiPSC-derived cardiomyocytes are largely mononucleated in vitro, reflecting a foetal-like phenotype rather than the binucleation seen in postnatal maturation [26]. This limitation is particularly relevant in the context of disease modelling, as cardiomyopathic phenotypes may only emerge at later developmental stages. McKay et al. [17] reported that cardiomyopathy-associated symptoms in Alms1-deficient mice became apparent only after 23 weeks, highlighting a delayed onset that may not be captured in immature hiPSC-derived models.

Interestingly, in silico analysis (Figure S1) revealed the ALMS1:c.10004G>C variant as a pathogenic or likely pathogenic variant in humans. Moreover, the variant was initially identified in a feline model, in which it has been associated with the development of isolated hypertrophic cardiomyopathy [19]. In parallel to this cellular approach, we found the feline *ALMS1:*c.7384G>C counterpart of the human *ALMS1:*c.10004G>C variant to be a Variant of Unknown Significance in cats, using the American College of Medical Genetics and Genomics guidelines [28], thus necessitating further analysis of the effect of this variant. Our findings in human hiPSC-derived cardiomyocytes suggest that the variant’s primary effect may be on the efficiency of cardiomyocyte formation rather than on the contractile or structural properties of mature cardiomyocytes. This highlights a potential stage-specific vulnerability during cardiac development that may contribute rather than be a determinant of disease manifestation in vivo. Thus, the ALMS1:c.10004G>C variant may function as a modifier rather than a primary causal allele, subtly influencing specific aspects of cardiomyocyte biology.

To our knowledge, two ALMS1-deficient hiPSC lines have been reported. Ji et al. (2020) generated an iPSC line from peripheral blood mononuclear cells of a patient with compound heterozygous nonsense mutations in ALMS1—ALMS1:c.3902C>A, ALMS1:p.(S1301X) and ALMS1:c.6436C>T, ALMS1:p.(R2146X)—without reporting the phenotypic characterisation of the line [29]. Recently, Patel et al. generated an ALMS1 KO line and identified an altered phenotype in their ALMS1-deficient hiPSC-CMs [7]. Contrary to what we observed for our ALMS1-mutant line, they reported no reduction in the efficiency of cardiomyocyte differentiation. They reported an increased contraction amplitude in the ALMS1-deficient cells compared to an isogenic control but no other contractility defect. Sarcomere organisation was not explored. The major phenotypic alterations they observed were related to calcium handling, metabolism (glycolytic rate), and senescence, which are relevant pathways to explore in more detail given the spectrum of clinical manifestations of Alström syndrome. The two hiPSC lines reported are relevant tools for exploring ALMS1 KO consequences. The third ALMS1 mutant line we generated and reported here was derived using a missense variant associated with a feline cardiomyopathy. Our study was exploratory and had several limitations. It primarily relied on image-based analyses, with limited functional characterisation of cardiomyocytes. The molecular and developmental assessments were restricted to selected markers and timepoints, and nuclear morphology analyses were performed on replated cardiomyocytes, which may not fully represent three-dimensional cultures. In addition, apoptosis was not explored as it was not a primary endpoint of the study. Thus, our conclusions are based on analyses restricted to viable, differentiated cardiomyocytes. The findings should therefore be interpreted as hypothesis-generating.

In conclusion, we generated and initiated the characterisation of a human iPSC line carrying the ALMS1:c.10004G>C variant in the homozygous state but further research is needed to fully describe its phenotype and its relevance in modelling ALMS1-related changes in cardiomyocytes. Furthermore, our results support the previous recommendation [28] not to use ALMS1:c.7384G>C for the selection of breeding cats until further data confirm its intrinsic pathogenicity.