Major ß cell-specific functions of NKX2.2 are mediated via the NK2-specific domain.

The consolidation of unambiguous cell fate commitment relies on the ability of transcription factors (TFs) to exert tissue-specific regulation of complex genetic networks. However, the mechanisms by which TFs establish such precise control over gene expression have remained elusive-especially in instances in which a single TF operates in two or more discrete cellular systems. In this study, we demonstrate that ß cell-specific functions of NKX2.2 are driven by the highly conserved NK2-specific domain (SD). Mutation of the endogenous NKX2.2 SD prevents the developmental progression of ß cell precursors into mature, insulin-expressing ß cells, resulting in overt neonatal diabetes. Within the adult ß cell, the SD stimulates ß cell performance through the activation and repression of a subset of NKX2.2-regulated transcripts critical for ß cell function. These irregularities in ß cell gene expression may be mediated via SD-contingent interactions with components of chromatin remodelers and the nuclear pore complex. However, in stark contrast to these pancreatic phenotypes, the SD is entirely dispensable for the development of NKX2.2-dependent cell types within the CNS. Together, these results reveal a previously undetermined mechanism through which NKX2.2 directs disparate transcriptional programs in the pancreas versus neuroepithelium.

Differential use of the Nkx2.2 NK2 domain in developing pancreatic islets and neurons.

The homeodomain transcription factor (TF) Nkx2.2 governs crucial cell fate decisions in several developing organs, including the central nervous system (CNS), pancreas, and intestine. How Nkx2.2 regulates unique targets in these different systems to impact their individual transcriptional programs remains unclear. In this issue of Genes & Development Abarinov and colleagues (pp. 490-504) generated and analyzed mice in which the Nkx2.2 SD is mutated and found that the SD is required for normal pancreatic islet differentiation but dispensable for most aspects of neuronal differentiation.

Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube.

Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis--memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM-sleep regulatory neurons.

The midbrain reticular formation (MRF) is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including sleep regulation. However, the molecular characteristics and development of MRF neurons are poorly understood. As the transcription factor, Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is required for the expression of several GABAergic lineage-specific transcription factors, including Nkx2-2 and Skor2, which are co-expressed in a restricted group of post-mitotic GABAergic precursors in the MRF. Both Gata2 and Nkx2-2 function is required for Skor2 expression in GABAergic precursors. In the adult mouse and rat midbrain, Nkx2-2-and Skor2-expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the MRF, an area containing REM-off neurons regulating REM sleep. In addition to the characteristic localization, Skor2+ cells increase their activity upon REM-sleep inhibition, send projections to the dorsolateral pons, a region associated with sleep control, and are responsive to orexins, consistent with the known properties of midbrain REM-off neurons.

The myelination-associated G protein-coupled receptor 37 is regulated by Zfp488, Nkx2.2, and Sox10 during oligodendrocyte differentiation.

Oligodendrocyte differentiation and myelination in the central nervous system are controlled and coordinated by a complex gene regulatory network that contains several transcription factors, including Zfp488 and Nkx2.2. Despite the proven role in oligodendrocyte differentiation little is known about the exact mode of Zfp488 and Nkx2.2 action, including their target genes. Here, we used overexpression of Zfp488 and Nkx2.2 in differentiating CG4 cells to identify aspects of the oligodendroglial expression profile that depend on these transcription factors. Although both transcription factors are primarily described as repressors, the detected changes argue for an additional function as activators. Among the genes activated by both Zfp488 and Nkx2.2 was the G protein-coupled receptor Gpr37 that is important during myelination. In agreement with a positive effect on Gpr37 expression, downregulation of the G protein-coupled receptor was observed in Zfp488- and in Nkx2.2-deficient oligodendrocytes in the mouse. We also identified several potential regulatory regions of the Gpr37 gene. Although Zfp488 and Nkx2.2 both bind to one of the regulatory regions downstream of the Gpr37 gene in vivo, none of the regulatory regions was activated by either transcription factor alone. Increased activation by Zfp488 or Nkx2.2 was only observed in the presence of Sox10, a transcription factor continuously present in oligodendroglial cells. Our results argue that both Zfp488 and Nkx2.2 also act as transcriptional activators during oligodendrocyte differentiation and cooperate with Sox10 to allow the expression of Gpr37 as a modulator of the myelination process.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing.

BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation. RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed. CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

Clinicopathological and molecular genetic analysis of 13 cases of primary retroperitoneal Ewing sarcoma.

Retroperitoneal Ewing sarcomas (RES) are very rare and mostly described in case reports. The purpose of this study was to retrospectively analyze the clinicopathology, molecular characteristics, biological behavior, and therapeutic information of 13 cases of primary RES with immunohistochemical staining, fluorescence in situ hybridization, RT-PCR and NGS sequencing detection techniques. The thirteen patients included eight males and five females with a mean age of 34 years. Morphologically, the tumors were comprised of small round or epithelial-like cells with vacuolated cytoplasm (6/13,46 %) arranged in diffuse, nested (8/13,62 %) and perivascular (7/13,54 %) patterns. Unusual morphologic patterns, such as meningioma-like swirling structures and sieve-like structures were relatively novel findings. Immunohistochemical studies showed CD99 (12/13; 92 %), CD56 (11/13; 85 %), NKX2.2 (9/13; 69 %), PAX7 (10/11;91 %) and CD117(6/9;67 %) to be positive.12 cases (92 %) demonstrated EWSR1 rearrangement and 3 cases displayed EWSR1::FLI1 fusion by FISH. ERCC4 splice-site variant, a novel pathogenic variant, was discovered for the first time via RNA sequencing. With a median follow-up duration of 14 months (6 to 79 months), 8/13 (62 %) patients died, while 5/13(38 %) survived. Three cases recurred, and five patients developed metastasis to the liver (2 cases), lung (2 cases) and bone (1 case). RES is an aggressive, high-grade tumor, prone to multiple recurrences and metastases, with distinctive morphologic, immunohistochemical, and molecular genetic features. ERCC4 splicing mutation, which is a novel pathogenic variant discovered for the first time, with possible significance for understanding the disease, as well as the development of targeted drugs.

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors.

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2-deficient mice exhibit autism-like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

Genomic analyses identify molecular subtypes of pancreatic cancer.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma.

Core regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and adolescents carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC 'trio' constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other's super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, CRC-downstream genes and signaling pathways may contain potential therapeutic targets for this malignancy.

A unique case of adamantinoma-like Ewing sarcoma in the calcaneus, exhibiting prominent squamous differentiation and displaying EWSR1 gene rearrangement.

Adamantinoma-like Ewing sarcoma is uncommonly reported in the skeletal sites, including small bones of the feet.A 15-year-old girl presented with pain and swelling in her left foot, leading to difficulty in walking for 8 months. Plain radiograph revealed an ill-defined, lytic-sclerotic lesion without significant periosteal reaction in her left calcaneus. Magnetic resonance imaging (MRI) revealed an expansile lesion involving the anterior calcaneus, which was hypointense on T1 and heterogeneously hyperintense on T2-weighted sequences, infiltrating the adjacent bones and soft tissues. On imaging, the differential diagnoses considered were a giant cell tumor and other primary bone tumors.Histopathological examination revealed a tumor composed of small round cells, with interspersed keratin pearls. Immunohistochemically, the tumor cells were positive for CD99/MIC2, pan-cytokeratin (AE1/AE3), p40, p63, NKX2.2, and synaptophysin. Diagnosis of adamantinoma-like Ewing sarcoma was offered on the initial biopsy. Furthermore, the tumor cells revealed EWSR1 gene rearrangement by fluorescence in situ hybridization, confirming this diagnosis. The patient underwent neoadjuvant chemotherapy, had a poor response, and finally underwent below-knee amputation.This constitutes a rare case of adamantinoma-like Ewing sarcoma in the calcaneus. Ewing sarcoma may be considered as a differential diagnosis for intraosseous lytic-sclerotic lesions, even without significant periosteal reaction, at unusual sites, such as the bones of the foot. Awareness of this entity and application of ancillary techniques is recommended for its exact diagnosis and in differentiating this rare variant from its diagnostic mimics. This case also indicates a poor chemotherapy response in this unusual variant of Ewing sarcoma, occurring in the calcaneus.

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in ~ 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.

The transcription factor NKX2-2 regulates oligodendrocyte differentiation through domain-specific interactions with transcriptional corepressors.

The homeodomain protein NK2 homeobox 2 (NKX2-2) is a transcription factor that plays a critical role in the control of cell fate specification and differentiation in many tissues. In the developing central nervous system, this developmentally important transcription factor functions as a transcriptional repressor that governs oligodendrocyte (OL) differentiation and myelin gene expression, but the roles of various NKX2-2 structural domains in this process are unclear. In this study, using in situ hybridization, immunofluorescence, and coimmunoprecipitation, we determined the structural domains that mediate the repressive functions of murine NKX2-2 and identified the transcriptional corepressors that interact with it in OL cells. Through in ovo electroporation in embryonic chicken spinal cords, we demonstrate that the N-terminal Tinman domain and C-terminal domain synergistically promote OL differentiation by recruiting distinct transcriptional corepressors, including enhancer of split Groucho 3 (GRG3), histone deacetylase 1 (HDAC1), and DNA methyltransferase 3 α (DNMT3A). We also observed that the NK2-specific domain suppresses the function of the C-terminal domain in OL differentiation. These findings delineate the distinct NKX2-2 domains and their roles in OL differentiation and suggest that NKX2-2 regulates differentiation by repressing gene expression via multiple cofactors and molecular mechanisms.

Rnf220 cooperates with Zc4h2 to specify spinal progenitor domains.

During early embryonic development of the spinal cord, graded sonic hedgehog signaling establishes distinct ventral progenitor domains by regulating the spatiotemporal expression of fate-specifying transcription factors. However, regulation of their protein stability remains incompletely understood. Here, we show that RNF220, an E3 ubiquitin ligase, plays crucial roles in the generation of the ventral progenitor domains, which produce ventral interneurons and motor neurons, by targeting key transcription factors including Dbx1/2 and Nkx2.2 for degradation. Surprisingly, RNF220 interacts with, and is co-expressed with, a zinc-finger protein ZC4H2, and they cooperate to degrade Dbx1/2 and Nkx2.2. RNF220-null mice show widespread alterations of ventral progenitor domains, including the loss of the p2 domain that produces V2 interneurons. Knockdown of RNF220 and ZC4H2 in the chick spinal cord downregulates expression of the V2 interneuronal marker Chx10. Co-expression of RNF220 and ZC4H2 further promotes the ability of Nkx6.1 to induce ectopic Chx10+ V2 interneurons. Our results uncover a novel regulatory pathway in establishing distinct progenitor domains through modulating the protein stability of transcription factors. Our results provide insights into the molecular mechanism by which ZC4H2 mutations lead to human syndromes characterized by delayed motor development.

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression of endocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.

Identification of EWSR1-NFATC2 fusion in simple bone cysts.

AIMS: Simple bone cysts are benign intramedullary tumours primarily involving the long bones in skeletally immature individuals. Several mechanisms have been proposed for their pathogenesis. Although the diagnosis is typically straightforward, the interpretation can be problematic, because of superimposed fracture causing them to resemble aneurysmal bone cysts and other tumours. EWSR1-NFATC2 or FUS-NFATC2 fusions, which are characteristic of a subset of aggressive round cell sarcomas, have been recently detected in simple bone cysts. The aim of this study was to examine the clinicopathological and molecular features in a series of simple bone cysts. METHODS AND RESULTS: Using RNA-based next-generation sequencing and/or fluorescence in-situ hybridisation, we investigated the presence of EWSR1 or FUS rearrangements in nine simple bone cysts. The patients were five females and four males, aged 3-23 years (median, 14 years); the tumours ranged from 19 mm to 160 mm (median, 46 mm) in size, and involved the femur (n = 3), humerus (n = 2), fibula (n = 2), tibia (n = 1), and iliac wing (n =1). We identified three cases with EWSR1-NFATC2 fusion (showing identical breakpoints to those in EWSR1-NFATC2 sarcomas) and one additional case with FUS rearrangement. Unlike in EWSR1-NFATC2 sarcomas, immunohistochemical expression of NKX3.1 and NKX2.2 was absent in two simple bone cysts tested. CONCLUSIONS: More than 40% of simple bone cysts harbour genetic alterations confirming that they are neoplastic, investigation of EWSR1 and/or FUS rearrangement may help to distinguish simple bone cysts from mimics, and NFATC2 rearrangement is not pathognomonic of malignancy.

NKX3.1 immunoreactivity is not identified in mesenchymal chondrosarcoma: a 25-case cohort study.

AIMS: Mesenchymal chondrosarcoma (MC) is characterised typically by a bimorphic microscopic appearance of islands of a well-differentiated cartilaginous component, admixed with a primitive small cell component, which commonly expresses CD99 and NKX2.2. Given the variable relative abundance of each component and histological overlap with other small round cell tumours, the diagnosis can be challenging, especially in a limited sample. A distinctive gene fusion between HEY1 (located in 8q21) and NCOA2 (located in 8q13) was identified in MC, but the downstream molecular events are unknown. NKX3.1 (coding gene located in 8p21.1) was recently reported to be expressed in a small number of MC cases. The purpose of this study was to evaluate the potential diagnostic utility of NKX3.1 immunohistochemistry in MC. METHODS AND RESULTS: We evaluated sections from 25 cases of MC, including 20 extraskeletal and five osseous. The tumour affected nine females and 16 males, with a median age of 34 years (age range = 7-82 years). Two different rabbit antibodies against NKX3.1 (monoclonal and polyclonal) were used for immunohistochemistry. However, no immunoreactivity was observed with either of the antibodies in all 25 (100%) MC. CONCLUSIONS: NKX3.1 immunoreactivity was not identified in our cohort. Clonality of the antibody could not explain the negativity.

Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population.

BACKGROUND: Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. METHODS: We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. RESULTS: Sixty-six patients with pre-biopsy MDC scores of 3-8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. CONCLUSIONS: We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.