Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria mitochondrial disorder: case report and literature review

Background: Leigh syndrome is a rare, genetic, and severe mitochondrial disorder characterized by neuromuscular issues (ataxia, seizure, hypotonia, developmental delay, dystonia) and ocular abnormalities (nystagmus, atrophy, strabismus, ptosis). It is caused by pathogenic variants in either mitochondrial or nuclear DNA genes, with an estimated incidence rate of 1 per 40,000 live births.Case presentation: Herein, we present an infant male with nystagmus, hypotonia, and developmental delay who carried a clinical diagnosis of Leigh-like syndrome. Cerebral magnetic resonance imaging changes further supported the clinical evidence of an underlying mitochondrial disorder, but extensive diagnostic testing was negative. Trio exome sequencing under a research protocol uncovered compound-heterozygous missense variants in the HTRA2 gene (MIM: #606441): NM_013247.5:c.1037A>T:(p.Glu346Val) (maternal) and NM_013247.5:c.1172T>A:(p.Val391Glu) (paternal). Both variants are absent from public databases, making them extremely rare in the population. The maternal variant is adjacent to an exon-intron boundary and predicted to disrupt splicing, while the paternal variant alters a highly conserved amino acid and is predicted to be damaging by nearly all in silico tools. Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria, type VIII (MGCA8), an extremely rare autosomal recessive disorder with fewer than ten families reported to date. Variant interpretation is challenging given the paucity of known disease-causing variants, and indeed we assess both paternal and maternal variants as Variants of Uncertain Significance under current American College of Medical Genetics guidelines. However, based on the inheritance pattern, suggestive evidence of pathogenicity, and significant clinical correlation with other reported MGCA8 patients, the clinical care team considers this a diagnostic result.Conclusion: Our findings ended the diagnostic odyssey for this family and provide further insights into the genetic and clinical spectrum of this critically under-studied disorder

Role of OCRL1-Dependent Signaling Abnormalities and Mutation Heterogeneity in Lowe Syndrome Cellular Phenotypes

Lowe Syndrome (LS) is a lethal developmental disease characterized by mental retardation, cataracts at birth and kidney dysfunction. LS children unfortunately die by adolescence from renal failure. The gene responsible for the disease (OCRL1) encodes an inositol 5’ phosphatase Ocrl1. In addition to its 5’ phosphatase domain, this protein has other domains that allow protein-protein interactions, facilitating diverse sub-cellular distribution and functions. LS patient cells lacking Ocrl1display defects in cell spreading, ciliogenesis and vesicle trafficking. Currently the mechanisms underlying these cellular defects are not known, and hence no LS-specific therapies exist. We have uncovered the mechanisms underlying two LS-specific cellular phenotypesnamely cell spreading and ciliogenesis and identified 2 FDA-approved candidates- statins and rapamycin that could revert these abnormalities. We found that Ocrl1-deficient cells exhibit hyperactivation in mTOR signaling, resulting in ciliogenesis as well as autophagy defects, which were rescued by administering rapamycin. We also identified a novel RhoGTPase signalingdependent cell adhesion defect in LS patient cells which resulted in focal adhesion abnormalities and sensitivity to fluid shear stress (critical for kidney function). Both RhoGTPase signaling dependent cell spreading and adhesion defects were corrected by treatment with statins. Importantly, over 200 unique mutations in OCRL1 cause LS and patients demonstrate heterogeneity in symptoms. However, the correlation between genotype and cellular phenotypes is unknown. We have determined that different OCRL1patient mutations have a differential impact on the two cellular phenotypes described above. Mutants exhibit behavior, sub-cellular distribution and cellular phenotypes unique to the domain and relevant to LS pathogenesis. We also propose that a subset of non-catalytic phosphatase domain mutations are conformationally affecting the protein, suggesting that LS has a conformational disease component. Importantly, we tested an FDA-approved drug, 4-phenyl butyric acid (4-PBA), used as a therapeutic in conformational diseases and found that it could revert phenotypes and restore the catalytic activity of these mutants. These findings collectively contribute to provide the cellular basis for LS patient heterogeneity as well as to propose a conformational disease component for LS (allowing the use of chemical chaperones as a therapeutic strategy for a subset of LS patients). Together, we hope that these studies will help lay the foundation of better prognosis and tailoring personalized therapeutic strategies for LS patients

Kidney-differentiated cells derived from Lowe Syndrome patient’s iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex

<div><p>Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the <i>OCRL1</i> gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient’s cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, <i>OCRL1</i> KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2’s role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.</p></div

LS renal cells show defects in primary cilia assembly.

<p>Ciliogenesis in kidney-differentiated cells was induced as described in Materials and methods. <b>A</b> and <b>C</b>. Primary cilia (PC) presence was investigated in renal-differentiated (<b>A</b>) and HK2 (<b>C</b>) cells by indirect immunofluorescence using anti-acetylated tubulin (Ac-Tub, green), anti-pericentrin-2 (PC2, red) antibodies. DAPI staining (blue) was performed to highlight nucleus position. Scale bar: 10μm. <b>B</b> and <b>D</b>. The percentage of cells displaying PC was determined for normal and LS renal differentiated (<b>B</b>) and HK2 (<b>D</b>) cells. Statistical significance of difference between means was assessed by using the student <i>t</i>-test (**: p<0.05).</p

HK2 <i>OCRL1</i>^-/- proximal tubule cells show Six2 retention in the Golgi complex.

<p>WT and <i>OCRL1</i> KO (-/-) cells were monitored for Six2 intracellular localization as described in Materials and methods as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192635#pone.0192635.g004" target="_blank">Fig 4A and 4B</a>. Statistical significance of difference between means was assessed by using the Wilcoxon test (**: p<0.05).</p

Generation of normal- and LS patient-derived iPSCs from skin fibroblasts.

<p><b>A.</b> iPSCs from normal and patient fibroblasts were prepared as described in Materials and methods. Cell and colony morphology differences were evident using bright-field microscopy, expression of the iPSC marker Tra-1-60 was investigated using direct immunofluorescence of live fibroblasts and iPSCs (see text for details). Scale bar: 100μm. <b>B</b> and <b>C</b>. Expression and localization of the transcription factor Oct4 (<b>B</b>) and adhesion molecule E-cadherin (ECAD, <b>C</b>) was investigated in normal/LS patient fibroblasts (<i>left</i>) and iPSCs (<i>right</i>) using indirect immunofluorescence with specific antibodies (red). DAPI staining (blue) was performed to highlight nucleus position. Scale bar: 10μm. <b>D</b>. Expression of several stem cell markers was investigated by quantitative RT-PCR as described in Materials and methods. Results were expressed as folds change observed in iPSC with respect to the corresponding fibroblast, results were also controlled with respect to RPLP0 expression. A representative experiment is shown.</p

Generation of normal and LS renal cell lineages.

<p><b>A</b>. iPSCs were differentiated as renal cells following the procedure described in Materials and methods. <b>B</b>. The presence of the kidney-specific marker Cadherin16 (Cad16) was investigated by Western blotting using a specific antibody. Tubulin was detected with a specific antibody and used as loading control. <b>C</b> and <b>D</b>. Expression and localization of the renal progenitor intermediate mesoderm marker Pax2 and mesendoderm marker N-cadherin (NCAD) was investigated in normal/LS patient differentiated cells using indirect immunofluorescence with specific antibodies (red). DAPI staining (blue) was performed to highlight nucleus position. Scale bar: 10μm.</p

The ductal lineage-inhibitor and cap mesenchyme marker Six2 is retained in the Golgi complex of LS cells.

<p><b>A</b>. Normal (upper panels) and LS (middle and lower panels) kidney-differentiated cells treated with NaCl or LiCl were immunostained using anti-Six2 (red) and anti-GM130 (green) antibodies, the location of the nucleus was revealed by DAPI staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192635#sec009" target="_blank">Materials and methods</a>). Arrows point to some examples of Six2-GM130 colocalization; arrowheads highlight examples of Six2 nuclear exclusion. Scale bar: 10μm. <b>B-C</b>. The fluorescence intensity of the Six2-signal associated with the Golgi complex (<b>B</b>) or the nucleus (<b>C</b>) was quantified and expressed as a fraction of the total Six2 fluorescence (B) or of the number of cells (C) in normal and LS kidney-differentiated cells treated with NaCl or LiCl as indicated. More than 50 cells from at least 5 independent determinations. Statistical significance of the difference between normal and LS samples was assessed using the Wilcoxon test (B) or the <i>t</i>-test (C). **: p<0.05. <b>D</b>. Representative images of Ck8-positive kidney-differentiated normal and LS cells. Note the increased Ck8⁺ fraction (total Ck8 positive cells/total cells) within LS kidney cells. <b>E</b>. Quantification of the relative fraction of LS to normal cells showing Six2 nuclear staining (left in red) and Ck8-positive cells (right in green) in the absence of NaCl or LiCl. In all cases, experiments were repeated using at least 5 differentiation batches (out of different iPSC clones).</p

Lowe syndrome patient cells display mTOR- and RhoGTPase-dependent phenotypes alleviated by rapamycin and statins

Lowe syndrome (LS) is an X-linked developmental disease characterized by cognitive deficiencies, bilateral congenital cataracts and renal dysfunction. Unfortunately, this disease leads to the early death of affected children often due to kidney failure. Although this condition was first described in the early 1950s and the affected gene (OCRL1) was identified in the early 1990s, its pathophysiological mechanism is not fully understood and there is no LS-specific cure available to patients. Here we report two important signaling pathways affected in LS patient cells. While RhoGTPase signaling abnormalities led to adhesion and spreading defects as compared to normal controls, PI3K/mTOR hyperactivation interfered with primary cilia assembly (scenario also observed in other ciliopathies with compromised kidney function). Importantly, we identified two FDA-approved drugs able to ameliorate these phenotypes. Specifically, statins mitigated adhesion and spreading abnormalities while rapamycin facilitated ciliogenesis in LS patient cells. However, no single drug was able to alleviate both phenotypes. Based on these and other observations, we speculate that Ocrl1 has dual, independent functions supporting proper RhoGTPase and PI3K/mTOR signaling. Therefore, this study suggest that Ocrl1-deficiency leads to signaling defects likely to require combinatorial drug treatment to suppress patient phenotypes and symptoms

Heterogeneity in Lowe Syndrome: Mutations Affecting the Phosphatase Domain of OCRL1 Differ in Impact on Enzymatic Activity and Severity of Cellular Phenotypes

Lowe Syndrome (LS) is a condition due to mutations in the OCRL1 gene, characterized by congenital cataracts, intellectual disability, and kidney malfunction. Unfortunately, patients succumb to renal failure after adolescence. This study is centered in investigating the biochemical and phenotypic impact of patient’s OCRL1 variants (OCRL1VAR). Specifically, we tested the hypothesis that some OCRL1VAR are stabilized in a non-functional conformation by focusing on missense mutations affecting the phosphatase domain, but not changing residues involved in binding/catalysis. The pathogenic and conformational characteristics of the selected variants were evaluated in silico and our results revealed some OCRL1VAR to be benign, while others are pathogenic. Then we proceeded to monitor the enzymatic activity and function in kidney cells of the different OCRL1VAR. Based on their enzymatic activity and presence/absence of phenotypes, the variants segregated into two categories that also correlated with the severity of the condition they induce. Overall, these two groups mapped to opposite sides of the phosphatase domain. In summary, our findings highlight that not every mutation affecting the catalytic domain impairs OCRL1′s enzymatic activity. Importantly, data support the inactive-conformation hypothesis. Finally, our results contribute to establishing the molecular and structural basis for the observed heterogeneity in severity/symptomatology displayed by patients