Published papers

Abstract Context Recent studies have revealed that every eukaryotic cell contains several membraneless organelles created via liquid-liquid phase separation (LLPS). LLPS is a physical phenomenon that transiently compartmentalizes the subcellular space and thereby facilitates various biological reactions. LLPS is indispensable for cellular functions; however, dysregulated LLPS has the potential to cause irreversible protein aggregation leading to degenerative disorders. To date, there is no systematic review on the role of LLPS in endocrinology. Evidence acquisition We explored previous studies which addressed roles of LLPS in living cells, particularly from the viewpoint of endocrinology. To this end, we screened relevant literature in PubMed published between 2009 and 2021 using LLPS-associated keywords including “membraneless organelle”, “phase transition”, and “intrinsically disordered”, and endocrinological keywords such as “hormone”, “ovary”, “androgen”, and “diabetes”. We also referred to the articles in the reference lists of identified papers. Evidence synthesis Based on 67 articles selected from 449 papers, we provided a concise overview of the current understanding of LLPS in living cells. Then, we summarized recent articles documenting the physiological or pathological roles of LLPS in endocrine cells. Conclusions The discovery of LLPS in cells has resulted in a paradigm shift in molecular biology. Recent studies indicate that LLPS contributes to male sex development by providing a functional platform for SOX9 and CBX2 in testicular cells. In addition, dysregulated LLPS has been implicated in aberrant protein aggregation in pancreatic β-cells, leading to type 2 diabetes. Still, we are just beginning to understand the significance of LLPS in endocrine cells.

Introduction: Kallmann syndrome (KS) is a genetically heterogeneous condition characterized by hypogonadotropic hypogonadism (HH) and olfactory dysfunction. Although SOX10, a causative gene for Waardenburg syndrome (WS) and peripheral demyelinating neuropathy, central demyelination, WS, and Hirschsprung disease (PCWH) has previously been implicated in KS, the clinical significance of SOX10 variants as the cause of KS remains uncertain. Patients and Methods: A total of 117 patients with KS underwent mutation screening of SOX10 and 14 other causative genes for KS/HH. Rare SOX10 variants were subjected to in silico and in vitro analyses. We also examined clinical data of the patients and their parents with SOX10 variants. Results: Sequence analysis identified 2 heterozygous variants of SOX10 (c.1225G > T, p.Gly409* and c.475C > T, p.Arg159Trp) in patients 1-3, as well as in the parents of patients 1 and 3. The variants were assessed as pathogenic/likely pathogenic, according to the American College of Medical Genomics guidelines. Both variants lacked in vitro transactivating activity for the MITF promoter and exerted no dominant-negative effects. Patients 1-3 carried no pathogenic variants in other genes examined. The patients presented with typical KS, while such features were absent in the parents of patients 1 and 3. None of the 5 variant-positive individuals exhibited hypopigmentation, while 1 and 2 individuals exhibited complete and partial hearing loss, respectively. Conclusion: These results provide evidence that SOX10 haploinsufficiency accounts for a small percentage of KS cases. SOX10 haploinsufficiency is likely to be associated with a broad phenotypic spectrum, which includes KS without other clinical features of WS/PCWH.

We describe a case of posthumously diagnosed MIRAGE syndrome (Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital problems, and Enteropathy) in a girl with a new pathogenic SAMD9 variant (p.F437S), who was initially considered to have familial dysautonomia (FD)-like disease due to increased levels of catecholamine metabolites. Functional analyses of F437S-SAMD9 were performed, showing characteristics of disease-causing variants. This new SAMD9 variant (p.F437S) also causes MIRAGE syndrome.

Membrane-less organelles (MLOs) are formed by biomolecular liquid-liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Alpha-thalassemia/mental retardation syndrome X-linked (ATRX; OMIM #301040), which is caused by mutations in the ATRX gene, is characterized by alpha-thalassemia, distinct dysmorphic facies, psychomotor development delay and genital abnormalities. Here, we describe a neonatal case of syndromic disorder of sex development, harboring a novel hemizygous mutation, p.Asp2352fs*1 in the carboxyl-terminal domain of ATRX. Our study provides additional evidence that deletion of the carboxyl terminus of ATRX is associated with severe genital anomalies.