Human Studies: All human studies were conducted in accordance with regulation of the University of California Los Angeles (UCLA) Institutional Review Board (IRB). Subjects provided written informed consent to participate in this study. Electronic medical record, family pedigree and specimen collection were acquired through the UCLA Congenital Heart Defect (CHD) BioCore  following the UCLA-IRB approved protocols. Specimens were de-identified and coded following acquisition.
Case of Interest: The pEFE proband is a female infant of healthy parents of Hispanic descent, who presented with profound congestive heart failure at 6 weeks of life. The prenatal course and delivery history were uneventful. Newborn screening, infectious work-up and comprehensive metabolic panel revealed no abnormalities [Figure 1. A and B]. pEFE diagnosis was determined based on clinical pathological examination of the explanted heart. No structural defect was identified. At the time of heart transplantation, the ejection fraction EF of the left ventricle was less than 20%. The clinical time course of the participant is summarized in Figure 1. C.
Pathological Findings: Pathological examination was performed by an expert anatomic pathologist. A gross image of the explanted pEFE heart is shown in Figure 1. D. Diffuse thickening of LV endocardium with an elevation of the papillary muscles and thickening of the free edges of mitral valve leaflets were observed, giving an opaque appearance to the endocardial surface. Compared to an age matched heart with dilated cardiomyopathy (DCM), the LV endocardial thickness is significantly greater in pEFE [Figure 1.E a&b] with dense elastic fibers arranged in a longitudinal pattern shown by Trichrome staining [Figure 1.E c&d]. In contrast, collagen IV deposits were less pronounced [Figure 1.E e&f]. Based on these pathological features, a clear distinction could be made between pEFE heart and DCM heart.
Potential Contribution of Recessive Ciliopathy Genes to pEFE Etiology:To investigate the genetic contribution to pEFE etiology, we performed WES on our pEFE proband and her parents combined with RNA-seq analysis of pEFE proband dermal fibroblasts [Supplemental Figure 1]. Parents received pre-test genetic counseling and were then asked to provide informed consent to make anonymous clinical and genomic data available for research and publication. Using a custom-made primary gene list of 44 known cardiomyopathy genes, including TAZ [Supplenmental Table 1], no pathogeneic variants were identified. Based on the sporadic nature of pEFE presentation in the proband cohort, we expected that de novo germline mutations might contribute to the genetic basis of pEFE. However, no denovo mutation was detected. The family pedigree did not reveal parental consanguinity or another affected family member. On the contrary, homozygosity analysis revealed 8 large runs of homozygosity (ROH) greater than 5 MBs, totaling 51.8 MBs, which correspond to 1.62% of the proband’s genome [Supplemental Table 2], suggesting that both parents descend from a common, genetically isolated community. Importantly, within these ROHs, three novel homozygous variants in cilia-related genes, DNAH8, DNAH17, and ALMS1, were detected, while both of the unaffected parents were heterozygous carriers for each of these variants [Figure 2. A]. Integrative Genomics Viewer (IGV) analysis confirmed the zygosity of the variants [Figure 2. B]. The detected variants have not been previously reported in gnomAD, ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), the 1000 Genomes database (http://www.internationalgenome.org), or ExAC (http://exac.broadinstitute.org/). By Sanger sequencing, we confirmed the newly identified homozygous variants using gDNA obtained from dermal fibroblasts derived from a punch skin biopsy of the pEFE proband, with human neonatal dermal fibroblasts (hNDFs) used as a negative control [Figure 2.C].
DNAH8 and DNAH17 genes encode two heavy chain proteins of the axonemal dynein that generate force through ATP hydrolyzing and microtubule binding. The novel homozygous variant in DNAH17 [17:76487553 c.6641C>G] replaces a serine residue for cysteine at the amino acid 2214 (p.Ser2214Cys) and is predicted to be deleterious, suggesting this variant may not be well tolerated. The novel homozygous single nucleotide deletion in DNAH8 [6:38840474 c.7153delT] results in a frame shift [p.Phe2385fs] that causes a premature termination of translation leading to a truncated protein that only contains the first 2384 amino acids of DNAH8 followed by an extra stretch of 23 amino acids. Therefore, the ATPase activity and microtubule-binding ability are predicted to be affected by the mutation. Variants in DNAH17 and DNAH8 have been implicated in spermatogenic failure [OMIM: 618643] , and have never been reported in association with cardiomyopathy. Therefore, these two genes are unlikely to contribute to pEFE phenotype.
Finally, ALMS1 encodes the centrosome and basal body associated protein 1, which functions in the formation and maintenance of cilia and in microtubule organization [12, 13]. The novel ALMS1 variant [2: 73675594 c.1938delA] is a single nucleotide deletion, which results in a frameshift [p.Val649fs] and introduction of a premature stop codon. Mutations in ALMS1 are known causal of AS [OMIM 203800] [12-15], a rare autosomal recessive ciliopathy, which has been associate with cardiomyopathy [16-20]. However, AS has not been associated with pEFE phenotype and our patient did not have other clinical features of AS at the time of WES studies.
Prioritizing the Candidate Genes for Functional Assessment: To further prioriterize the candidate novel variants in the three cilia-related genes, we set out first to confirm the impact of these variants on RNA expression. We isolated total RNAs from pEFE proband dermal fibroblasts and from hNDFs (as a control) and performed paired-end RNA-seq as we previously described [23, 24]. Out of the three candidate genes, ALMS1 was the only gene expressed in dermal fibroblasts with good read coverage as illustrated by IGV viewer, while neither DNAH8 nor DNAH17 was detected. IGV confirmed the impact of the novel ALMS1 c.1938delA variant at the RNA level in pEFE fibroblast compared to control hNDF [Figure 2. D]. Next, we examined the expression of these genes in our previously reported RNA-seq data of congenital heart defects samples obtained from infants with structural heart defects . Among the three genes, only ALMS1 was expressed in heart tissue samples [Figure 2. E].
Given that the cardiac dysfunction of pEFE manifests clinically during the early postnatal period , we next examined the expression of the three candidate genes in our previously reported RNA-seq data derived from neonatal mouse heart left and right ventricles at postnatal day P0, P3 and P7 , available in the NCBI’s Gene Expression Omnibus repository under the Neonatal Heart Maturation SuperSeries [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85728]. Again, among the three candidate genes, ALMS1 was the only gene expressed in both ventricular chambers, exhibiting dynamic regulation in neonatal heart during perinatal stages [Figure 2. F]. To further ascertain the expression pattern of the candidate genes in neonatal heart at the cellular level, we examined their expression in primary cultured neonatal rat ventricular myocytes (NRVMs) and neonatal rat cardiac fibroblasts (NRCFs) isolated from neonatal rat hearts. Indeed, out of the three candidate genes, only ALMS1 was expressed in both cardiac cell types [Figure 2. G]. Altogether, our findings led to prioritizing ALMS1, which is expressed in neonatal heart and has been associated with cardiomyopathy, as the potential causal candidate gene contributing to pEFE. Therefore, we focused the following studies on determining the molecular and cellular impact of the novel ALMS1 variant in pEFE pathogenesis.
ALMS1 Protein Expression is Absent in pEFE Proband Dermal Fibroblasts: ALMS1 gene is located on chromosome 2p13.1 spanning 23 exons. It encodes a protein of 4,169 amino acids (461.2 kDa), which lacks known catalytic domains [25, 26], but has several sequence features of unknown function, including a large tandem repeat domain (TRD), three short coiled-coil domains and a stretch of ~130 residues at the C-terminus termed as the ALMS motif [aa: 4032-4164 (Ensembl)] [Figure 3. A]. The ALMS1 protein is widely expressed and co-localizes with the centrosomes and basal bodies of ciliated cells in different tissues and organs, where ALMS1 has been shown to play important roles in ciliary function and intracellular trafficking . Deletion analysis of ALMS1 suggests that the ALMS motif may play a key role in centrosome-targeting . Importantly, the variant of interest [c.1938delA] is predicted to result in a severely truncated ALMS1 protein [p.Val649fs], missing most of the tandem repeats and the highly conserved ALMS motif at the C-terminus. Based on these previously reported observations, we predicted the ALMS1 protein expression and/or stability in the centrioles to be affected by the novel ALMS1 variant in pEFE cells. Indeed, immunocytochemistry (ICC) assay using anti-ALMS1 antibodies confirmed ALMS1 protein localization to the centrioles in control hNDFs, while ALMS1 protein was completely absent in the proband pEFE-derived dermal fibroblasts, indicating a null effect [Figure 3. B].
Novel ALMS1 Variant Alters the Functional Phenotype of pEFE Fibroblasts: To examine the functional impact of the novel ALMS1 variant at the cellular level, we first examined cilia morphology in pEFE dermal fibroblasts compared to control hNDFs using electron microscopy (EM). We observed a unipolar organization of thin, over-branched, microvilli, nonmotile finger-like protrusions from the surface of epithelial cells that function to increase the adhesion, the cell surface area, the efficiency of absorption, that was more prominent in pEFE cells compared to control hNDFs [Figure 3. C]. Next we examined the impact on cellular proliferation over time using an automated proliferation assay performed on an xCELLigence RTCA SP instrument. We observed decreased proliferation index in pEFE fibroblasts, compared to hNDFs [Figure 3. Dand E]. FACS analysis revealed no change in G2/M phase in pEFE fibroblast compared to control [Supplemental Figure 2]. Then, we examined the impact on cellular migration over time using the xCELLigence RTCA DP instrument and observed increased migration activity in pEFE fibroblasts compared to the control hNDFs [Figure 3. F and G]. Together, the novel ALMS1 variant altered the functional phenotype and cellular physiology of pEFE fibroblasts. To further confirm the impact of ALMS1 loss, we treated primary cultured hNDF1 with ALMS1-siRNA. Indeed, ALMS1-depleted hNDFs exhibited enhanced migration replicating the changes seen in pEFE fibroblasts [Supplemental Figure 3].
Novel ALMS1 Variant Impact on pEFE Proband Heart: As previously stated, few reports have implicated homozygous ALMS1 mutations in an extremely rare form of neonatal cardiomyopathy associated with AS and characterized by prolonged mitotic window of postnatal cardiomyocytes, hence termed “mitogenic cardiomyopathy” [16, 17]. Furthermore, mice with Alms1 mutations exhibit delayed exit from active cell cycle progression , supporting a functional role for ALMS1 in regulating postnatal cardiomyocyte maturation. Based on these reported observations, we sought to determine the pathogenic impact of the mutant ALMS1 in pEFE heart. First, we determined that ALMS1 protein is also localized at the centrosomic poles (centrioles) in neonatal cardiomyocytes by using primary cultured neonatal rat ventricular myocytes (NRVMs) isolated from the neonatal rat heart [Figure 4. A]. Next, we confirmed that ALMS1 loss is sufficient to extend the proliferative activity of postnatal cardiomyocytes by using small interfering RNA (siRNA)-mediated inhibition of Alms1 in primary cultured NRVMs. We observed increased mitotic activity in Alms1-deficient myocytes, as indicated by increased number of phospho-Histone H3 (pH3) positive cardiomyocytes and upregulation of the mitotic marker genes, including Ki67 and Cdc25c, compared to control scramble [Figure 4. B and C]. These results are consistent with previous studies [16-18], indicating that ALMS1 regulates neonatal cardiomyocyte proliferation. Then, we confirmed that ALMS1 expression at the centrosomes was absent in histopathological sections obtained from the proband pEFE heart, but appeared normal in the age matched DCM heart with a pathogenic mutation in TNNT2 [Figure 4. D]. Finally, we evaluated cardiomyocyte proliferation activity in proband pEFE heart sections, using pH3 immunohistochemistry (IHC) analysis and revealed increased pH3 positive cardiomyocytes in pEFE heart compared to the age-matched DCM heart [Figure 4. E and F]. Together, in agreement with other reported cases of AS-associated mitogenic cardiomyopathy, the novel ALMS1 variant abolished ALMS1 protein expression and localization at the centrioles and delayed cardiomyocyte proliferation arrest in pEFE heart. However, how novel ALMS1 vaiant leads to pEFE phenotype remains unclear.
Novel ALMS1 Variant Alters Global Transcriptome Signature in pEFE Fibroblasts: Having established the causal role of the novel ALMS1 variant [c.1938delA] in pEFE, we aimed to gain further insights into the mechanisms that underlie ALMS1 function in endocardial fibroelastosis process by evaluating the global impact of ALMS1 loss on transcriptome programming of proband pEFE cells that carry the mutation. We systematically analyzed pEFE dermal fibroblast-derived RNA-seq datasets using total RNA obtained from three independent biological replicates. In addition, three independent hNDF replicates were subjected to the same RNA-seq protocol as we previously described [23, 24].
Principal component analyses of the top 1000 varied genes showed that transcripts from pEFE and control fibroblasts formed distinct clusters [Figure 5. A]. Likewise, expression heatmap across the entire transcriptome revealed distinct molecular signatures for pEFE and control samples [Figure 5. B], indicating that the variation pattern was consistent across the two methods. In total, 8970 protein coding genes were expressed at ≥3 RPKM in at least three sample (3 biological replicates) with coefficient of variation (CV) exceeding 0.2. Of these expressed genes, 3910 genes exhibited significant differential gene expression (DGE) in pEFE versus control at Benjamini–Hochberg (B-H) adjusted P value less than 0.05 [Figure 5. C]. Together, these findings indicate significant impact of ALMS1 perturbation on global transcriptome signature in pEFE fibroblasts compared to the control hNDF cells.
Novel ALMS1 Variant Induces TGFβ Signaling and Activates Epithelial Mesenchymal Transition (EMT): Gene ontology analysis of the differentially expressed genes was performed using Gene Set Enrichment Analysis (GSEA) . EMT, a process that converts epithelial cells to mobile mesenchymal cells and plays an important role in cardiac development , was predominantly enriched in the upregulated genes. Altogether, 175 EMT-related genes exhibited significant induction [Supplemental Tables 3-5], including activated myofibroblast cell surface marker genes (EDA, POSTN), cell adhesion genes (ITGB3, ITGA2), extracellular matrix (ECM) genes (COL5A3, ELN), key transcription regulators of EMT (ID1, ID2, SNAIL), and major signaling players of EMT (FZD8, TGFβ). Importantly, consistent with acquiring enhanced motility and migration abilities, genes involved in ECM degradation (MMP14, LOXL1) and motility (FGFR2, PRKG2) were also upregulated. On the other hand, apical junction (JUP, CDSN), cytoskeletal organization (NEXN, ABLIM1), P53 signaling (PIDD1, CASP1), intercellular trafficking (SLC16A6, SLC29A2) and cell cycle genes were downregulated in pEFE4 fibroblast compared to control. Together, the molecular signature of pEFE fibroblasts is consistent with a migratory, non-proliferative, EMT induced cellular phenotype.
To further annotate the enriched functional identity and signaling pathways in the differentially expressed genes, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and identified cellular adhesion, exon guidance, TGFβ and RAP1 signaling as the top enriched functional pathways that were also interconnected by sharing several overlapping genes [Figure 5. D and E]. For example, genes involved in EMT significantly overlapped with cell adhesion molecule and TGFβ pathways. Among these hub genes, we identified BMPR1B, a known member of the bone morphogenetic protein (BMP) family of transmembrane serine/threonine kinases that has been associate with left ventricular mass . The ligands of this receptor are members of the BMPs and TGF-β superfamily. Consistently, upstream analysis using IPA (Ingenuity Pathway Analysis) of the connected networks predicted TGFβ as the top upstream regulator with remarkable induction of its downstream signaling mediators, including BMPR1B, ID1-4, SMAD9, INHBB, CHRD, and BMP6, many of which are key players of EMT during embryonic development and cardiogenesis  [Supplemental Tables 3-5]. Together, our findings suggested TGFβ mediated activation of EMT program in the proband pEFE fibroblasts.
To validate these findings, we examined the impact of the novel ALMS1 variant on selected EMT marker genes in primary cultured pEFE fibroblasts compared to control hNDFs using qRT-PCR. Indeed, TGFβ was induced along with the transcription factor SNAIL, the ECM marker ELN as well as the myofibroblast marker gene aSMA. Consistently, CDH1, a key signaling mediator for the induction of EMT cascade, was significantly upregulated, while CDH2 was downregulated. Finally, cell cycle markers (Ki67 and Plk1) were suppressed [Figure 6. A]. Together, the data support that ALMS1 loss leads to EMT induction, potentially via the activation of TGFβ signaling.
Novel ALMS1 Mutation Induces EMT in Neonatal Cardiac Fibroblasts: Current evidence suggests a role for EMT in EFE [30, 31]. Our transcriptome analysis suggested that ALMS1 loss altered the physiological proprieties (enhanced migration) and induced EMT in pEFE fibroblasts potentially via activating TGFβ signaling. We sought next to determine whether ALMS1 regulates EMT in cardiac fibroblasts by performing siRNA-mediated knockdown of Alms1 in primary cultured NRCFs. Remarkably, the cells exhibited enhanced migration and induced EMT marker genes, including the Snail1, Tgfb, and Cdh1 [Figure 6. B-D]. Importantly, unlike neonatal cardiomyocytes, NRCFs remained quiescent and did not exhibit increased proliferation activity as demonstrated by proliferation marker genes (Plk1 and Ki67) expression [Figure 6. D]. Together, the data suggest EMT induction and Tgfb activation in ALMS-deficient neonatal cardiac fibroblasts. These findings replicate the changes observed in the proband pEFE dermal fibroblasts and ALMS1-deficient hNDFs, and correspond to a known role of cilia-mediated signaling in driving EMT process in cardiac development and fibrosis in response to cardiac injury .
Further Surveillance for Alstrom Syndrome and Patient Care: According to the diagnostic criteria from Marshall et al (2007) , detecting the novel ALMS1 variant [c.1938delA] suggested the genetic diagnosis of AS. We provided detailed genetic consultation about AS to the family and monitored our pEFE proband closely for potential multisystem involvement, in addition to the regular monitoring and management by the primary cardiologist and heart transplantation clinic. Bilateral nystagmus, ptosis and photophobia were documented at 6-12 month of age. Visual impairment was diagnosed at one year of age. Standard electroretinography (ERG) at 7 years of age showed absence of scotopic, photopic, maximal-combined and flicker responses, consistent with severe pan-retinal abnormalities of both rod- and cone- mediated retinal functions [Figure 7. A]. Retinal imaging revealed bilateral retinal dystrophy [Figure 7. B]. Short stature, increased weight gain and early onset obesity were observed at 2-3 years of age associated with elevated triglycerids, acanthosis nigricans and increased hemoglobin A1C (HbA1C) suggesting insulin resistance [Figure 7. C-E]. Hearing evaluation, thyroid function tests, blood sugar levels, serum lipid profile, liver function tests, serum creatinine, abdominal ultrasound and blood chemistry remained normal. The patient maintained normal bilateral audiology and psychomotor functions.