Lymphatic endothelial cell-specific NRAS p.Q61R mutant embryos show abnormal lymphatic vessel morphogenesis.

Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.

Genetic backgrounds and genotype-phenotype relationships in anthropometric parameters of 116 Japanese individuals with Noonan syndrome.

Noonan syndrome (NS) is caused by pathogenic variants in genes encoding components of the RAS/MAPK pathway and presents with a number of symptoms, including characteristic facial features, congenital heart diseases, and short stature. Advances in genetic analyses have contributed to the identification of pathogenic genes in NS as well as genotype-phenotype relationships; however, updated evidence for the detection rate of pathogenic genes with the inclusion of newly identified genes is lacking in Japan. Accordingly, we examined the genetic background of 116 individuals clinically diagnosed with NS and the frequency of short stature. We also investigated genotype-phenotype relationships in the context of body mass index (BMI). Genetic testing revealed the responsible variants in 100 individuals (86%), where PTPN11 variants were the most prevalent (43%) and followed by SOS1 (12%) and RIT1 (9%). The frequency of short stature was the lowest in subjects possessing RIT1 variants. No genotype-phenotype relationships in BMI were observed among the genotypes. In conclusion, this study provides evidence for the detection rate of pathogenic genes and genotype-phenotype relationships in Japanese patients with NS, which will be of clinical importance for accelerating our understanding of the genetic backgrounds of Japanese patients with NS.

Comprehensive Analysis of a Japanese Pedigree with Biallelic ACAGG Expansions in RFC1 Manifesting Motor Neuronopathy with Painful Muscle Cramps.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive multisystem neurologic disorder caused by biallelic intronic repeats in RFC1. Although the phenotype of CANVAS has been expanding via diagnostic case accumulation, there are scant pedigree analyses to reveal disease penetrance, intergenerational fluctuations in repeat length, or clinical phenomena (including heterozygous carriers). We identified biallelic RFC1 ACAGG expansions of 1000 ~ repeats in three affected siblings having sensorimotor neuronopathy with spinocerebellar atrophy initially presenting with painful muscle cramps and paroxysmal dry cough. They exhibit almost homogeneous clinical and histopathological features, indicating motor neuronopathy. Over 10 years of follow-up, painful intractable muscle cramps ascended from legs to trunks and hands, followed by amyotrophy and subsequent leg pyramidal signs. The disease course combined with the electrophysical and imagery data suggest initial and prolonged hyperexcitability and the ensuing spinal motor neuron loss, which may progress from the lumbar to the rostral anterior horns and later expand to the corticospinal tract. Genetically, heterozygous ACAGG expansions of similar length were transmitted in unaffected family members of three successive generations, and some of them experienced muscle cramps. Leukocyte telomere length assays revealed comparatively shorter telomeres in affected individuals. This comprehensive pedigree analysis demonstrated a non-anticipating ACAGG transmission and high penetrance of manifestations with a biallelic state, especially motor neuronopathy in which muscle cramps serve as a prodromal and disease progress marker. CANVAS and RFC1 spectrum disorder should be considered when diagnosing lower dominant motor neuron disease, idiopathic muscle cramps, or neuromuscular hyperexcitability syndromes.

Nuclear pore pathology underlying multisystem proteinopathy type 3-related inclusion body myopathy.

OBJECTIVE: Multisystem proteinopathy type 3 (MSP3) is an inherited, pleiotropic degenerative disorder caused by a mutation in heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which can affect the muscle, bone, and/or nervous system. This study aimed to determine detailed histopathological features and transcriptomic profile of HNRNPA1-mutated skeletal muscles to reveal the core pathomechanism of hereditary inclusion body myopathy (hIBM), a predominant phenotype of MSP3. METHODS: Histopathological analyses and RNA sequencing of HNRNPA1-mutated skeletal muscles harboring a c.940G > A (p.D314N) mutation (NM_031157) were performed, and the results were compared with those of HNRNPA1-unlinked hIBM and control muscle tissues. RESULTS: RNA sequencing revealed aberrant alternative splicing events that predominantly occurred in myofibril components and mitochondrial respiratory complex. Enrichment analyses identified the nuclear pore complex (NPC) and nucleocytoplasmic transport as suppressed pathways. These two pathways were linked by the hub genes NUP50, NUP98, NUP153, NUP205, and RanBP2. In immunohistochemistry, these nucleoporin proteins (NUPs) were mislocalized to the cytoplasm and aggregated mostly with TAR DNA-binding protein 43 kDa and, to a lesser extent, with hnRNPA1. Based on ultrastructural observation, irregularly shaped myonuclei with deep invaginations were frequently observed in atrophic fibers, consistent with the disorganization of NPCs. Additionally, regarding the expression profiles of overall NUPs, reduced expression of NUP98, NUP153, and RanBP2 was shared with HNRNPA1-unlinked hIBMs. INTERPRETATION: The shared subset of altered NUPs in amyotrophic lateral sclerosis (ALS), as demonstrated in prior research, HNRNPA1-mutated, and HNRNPA1-unlinked hIBM muscle tissues may provide evidence regarding the underlying common nuclear pore pathology of hIBM, ALS, and MSP.

LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and controlling KLHL12-mediated collagen secretion.

Leucine zipper-like transcriptional regulator 1 (LZTR1), a substrate adaptor of Cullin 3 (CUL3)-based E3 ubiquitin ligase, regulates proteostasis of the RAS subfamily. Mutations in LZTR1 have been identified in patients with several types of cancer. However, the role of LZTR1 in tumor metastasis and the target molecules of LZTR1, excluding the RAS subfamily, are not clearly understood. Here, we show that LZTR1 deficiency increases tumor growth and metastasis. In lung adenocarcinoma cells, LZTR1 deficiency induced the accumulation of the RAS subfamily and enhanced cell proliferation, invasion, and xenograft tumor growth. Multi-omics analysis to clarify the pathways related to tumor progression showed that MAPK signaling, epithelial-mesenchymal transition (EMT), and extracellular matrix (ECM) remodeling-related gene ontology terms were enriched in LZTR1 knockout cells. Indeed, LZTR1 deficiency induced high expression of EMT markers under TGF-ß1 treatment. Our search for novel substrates that interact with LZTR1 resulted in the discovery of a Kelch-like protein 12 (KLHL12), which is involved in collagen secretion. LZTR1 could inhibit KLHL12-mediated ubiquitination of SEC31A, a component of coat protein complex II (COPII), whereas LZTR1 deficiency promoted collagen secretion. LZTR1-RIT1 and LZTR1-KLHL12 worked independently regarding molecular interactions and did not directly interfere with each other. Further, we found that LZTR1 deficiency significantly increases lung metastasis and promotes ECM deposition around metastatic tumors. Since collagen-rich extracellular matrix act as pathways for migration and facilitate metastasis, increased expression of RAS and collagen deposition may exert synergistic or additive effects leading to tumor progression and metastasis. In conclusion, LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and promoting collagen secretion. The functional inhibition of KLHL12 by LZTR1 provides important evidence that LZTR1 may be a repressor of BTB-Kelch family members. These results provide clues to the mechanism of LZTR1-deficiency carcinogenesis.

Neonatal developmental and epileptic encephalopathy with movement disorders and arthrogryposis: A case report with a novel missense variant of SCN1A.

Variants of SCN1A represent the archetypal channelopathy associated with several epilepsy syndromes. The clinical phenotypes have recently expanded from Dravet syndrome. CASE REPORT: We present a female patient with the de novo SCN1A missense variant, c.5340G > A (p. Met1780Ile). The patient had various clinical features with neonatal onset SCN1A epileptic encephalopathy, arthrogryposis multiplex congenita, thoracic hypoplasia, thoracic scoliosis, and hyperekplexia. CONCLUSION: Our findings are compatible with neonatal developmental and epileptic encephalopathy with movement disorders and arthrogryposis; the most severe phenotype probably caused by gain-of-function variant of SCN1A. The efficacy of sodium channel blocker was also discussed. Further exploration of the phenotype-genotype relationship of SCN1A variants may lead to better pharmacological treatments and family guidance.

CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Mecom mutation related to radioulnar synostosis with amegakaryocytic thrombocytopenia reduces HSPCs in mice.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome characterized by the congenital fusion of the forearm bones. RUSAT is largely caused by missense mutations that are clustered in a specific region of the MDS1 and EVI1 complex locus (MECOM). EVI1, a transcript variant encoded by MECOM, is a zinc finger transcription factor involved in hematopoietic stem cell maintenance that induce leukemic transformation when overexpressed. Mice with exonic deletions in Mecom show reduced hematopoietic stem and progenitor cells (HSPCs). However, the pathogenic roles of RUSAT-associated MECOM mutations in vivo have not yet been elucidated. To investigate the impact of the RUSAT-associated MECOM mutation on the phenotype, we generated knockin mice harboring a point mutation (translated into EVI1 p.H752R and MDS1-EVI1 p.H942R), which corresponds to an EVI1 p.H751R and MDS1-EVI1 p.H939R mutation identified in a patient with RUSAT. Homozygous mutant mice died at embryonic day 10.5 to 11.5. Heterozygous mutant mice (Evi1KI/+ mice) grew normally without radioulnar synostosis. Male Evi1KI/+ mice, aged between 5 and 15 weeks, exhibited lower body weight, and those aged ≥16 weeks showed low platelet counts. Flow cytometric analysis of bone marrow cells revealed a decrease in HSPCs in Evi1KI/+ mice between 8 and 12 weeks. Moreover, Evi1KI/+ mice showed delayed leukocyte and platelet recovery after 5-fluorouracil-induced myelosuppression. These findings suggest that Evi1KI/+ mice recapitulate the bone marrow dysfunction in RUSAT, similar to that caused by loss-of-function Mecom alleles.

Management of patients with presumed germline pathogenic variant from tumor-only genomic sequencing: A retrospective analysis at a single facility.

Cancer treatment is increasingly evolving toward personalized medicine, which sequences numerous cancer-related genes and identifies therapeutic targets. On the other hand, patients with germline pathogenic variants (GPV) have been identified as secondary findings (SF) and oncologists have been urged to handle them. All SF disclosure considerations for patients are addressed and decided at the molecular tumor boards (MTB) in the facility. In this study, we retrospectively summarized the results of all cases in which comprehensive genomic profiling (CGP) test was conducted at our hospital, and discussed the possibility of presumed germline pathogenic variants (PGPV) at MTB. MTB recommended confirmatory testing for 64 patients. Informed consent was obtained from attending physicians for 53 of them, 30 patients requested testing, and 17 patients tested positive for a confirmatory test. Together with already known variants, 4.5 % of the total confirmed in this cohort. Variants verified in this study were BRCA1 (n = 12), BRCA2 (n = 6), MSH2 (n = 2), MSH6 (n = 2), WT1 (n = 2), TP53, MEN1, CHEK2, MLH1, TSC2, PTEN, RB1, and SMARCB1. There was no difference in the tumor's VAF between confirmed positive and negative cases for variants determined as PGPV by MTB. Current results demonstrate the actual number of cases until confirmatory germline test for patients with PGPV from tumor-only CGP test through the discussion at the MTB. The practical results at this single facility will serve as a guide for the management of the selection and distribution of SF in the genome analysis.

Clinical decisions by the molecular tumor board on comprehensive genomic profiling tests in Japan: A retrospective observational study.

BACKGROUND: A paradigm shift has occurred in cancer chemotherapy from tumor-specific treatment with cytotoxic agents to personalized medicine with molecular-targeted drugs. Thus, it is essential to identify genomic alterations and molecular features to recommend effective targeted molecular medicines regardless of the tumor site. Nevertheless, it takes considerable expertise to identify treatment targets from primary-sequencing data in order to provide drug recommendations. The Molecular Tumor Board (MTB) denotes a platform that integrates clinical and molecular features for clinical decisions. METHODS: This study retrospectively analyses all the cases of discussion and decision at the MTB in Tohoku University Hospital and summarizes genetic alterations and treatment recommendations. RESULTS: The MTB discussed 1003 comprehensive genomic profiling (CGP) tests conducted in patients with solid cancer, and the resulting rate of assessing treatment recommendations was approximately 19%. Among hundreds of genes in the CGP test, only 30 genetic alterations or biomarkers were used to make treatment recommendations. The leading biomarkers that led to treatment recommendations were tumor mutational burden-high (TMB-H) (n = 32), ERBB2 amplification (n = 24), BRAF V600E (n = 16), and BRCA1/2 alterations (n = 32). Thyroid cancer accounted for most cancer cases for which treatment recommendation was provided (81.3%), followed by non-small cell lung cancer (42.4%) and urologic cancer (31.3%). The number of tests performed for gastrointestinal cancers was high (n = 359); however, the treatment recommendations for the same were below average (13%). CONCLUSION: The results of this study may be used to simplify treatment recommendations from the CGP reports and help select patients for testing, thereby increasing the accuracy of personalized medicine.

Reduced-intensity conditioning is effective for allogeneic hematopoietic stem cell transplantation in infants with MECOM-associated syndrome.

Mutations in the MECOM encoding EVI1 are observed in infants who have radioulnar synostosis with amegakaryocytic thrombocytopenia. MECOM-associated syndrome was proposed based on clinical heterogeneity. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for progressive bone marrow failure. However, data regarding allogeneic HSCT for this rare disease are limited. We retrospectively assessed overall survival, conditioning regimen, regimen-related toxicities and long-term sequelae in six patients treated with allogeneic HSCT. All patients received a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, cyclophosphamide or melphalan, and rabbit anti-thymocyte globulin and/or low-dose total body/thoracic-abdominal/total lymphoid irradiation, followed by allogeneic bone marrow or cord blood transplantation from unrelated donors between 4 and 18 months of age. All patients survived and achieved stable engraftment and complete chimerization with the donor type. Moreover, no patient experienced severe regimen-related toxicities, and only lower grades of acute graft-versus-host disease were observed. Three patients treated with low-dose irradiation had relatively short stature compared to three patients not treated with irradiation. Therefore, allogeneic HSCT with RIC is an effective and feasible treatment for infants with MECOM-associated syndrome. Future studies are needed to evaluate the use of low-dose irradiation to avoid risks of other long-term sequelae.

A novel variant in the transmembrane 4 domain of ANO3 identified in a two-year-old girl with developmental delay and tremor.

ANO3 encodes Anoctamin-3, also known as TMEM16C, a calcium-activated chloride channel. Heterozygous variants of ANO3 can cause dystonia 24, an adult-onset focal dystonia. Some pediatric cases have been reported, but most patients were intellectually normal with some exceptions. Here, we report a two-year-old girl who showed mild to moderate developmental delay, tremor, and ataxic gait, but no obvious dystonia. Trio exome sequencing identified a heterozygous de novo missense variant NM_031418.4:c.1809T>G, p.(Asn603Lys) in the ANO3 gene. Three cases with ANO3 variants and intellectual disability have been reported, including the present case. These variants were predicted to face in the same direction on the same alpha-helix (the transmembrane 4 domain), suggesting an association between these variants and childhood-onset movement disorder with intellectual disability. In pediatric cases with developmental delay and movement disorders such as tremor and ataxia, specific variants in the transmembrane 4 domain of ANO3 may be a cause, even in the absence of dystonia.

Comprehensive genomic profiling of a unique liposarcoma arising in a patient with Li-Fraumeni syndrome and the novel detection of c-myc amplification: a case report.

BACKGROUND: Germline TP53 mutations have been frequently reported in patients with Li-Fraumeni syndrome (LFS), resulting in a predisposition to various malignancies. Mutations other than germline TP53 mutations can also cause LFS-associated malignancies, but their details remain unclear. We describe a novel c-myc amplification in a unique liposarcoma in a patient with LFS. CASE PRESENTATION: A female patient with LFS developed breast cancer twice at the age of thirty; both were invasive ductal carcinomas harboring HER2 amplifications. Computed tomography revealed an anterior mediastinal mass, which was surgically resected. Histological analysis revealed three different lesions corresponding to myxoid liposarcoma-, pleomorphic liposarcoma-, and well-differentiated liposarcoma-like lesions. Fluorescence in-situ hybridization (FISH) analysis did not detect MDM2 amplification, Rb1 deletion, break apart signals of EWS, FUS, DDIT3, or c-myc, or c-myc-IGH fusion signals, but it did detect more c-myc signals. Further FISH analysis and comprehensive genomic profiling revealed c-myc amplification. We considered two differential diagnoses, dedifferentiated liposarcoma lacking MDM2 amplification and myxoid pleomorphic liposarcoma (MPLPS), and determined that this case is most likely MPLPS. However, definite diagnosis could not be made because a clear-cut differentiation of the case from liposarcomas was not possible. CONCLUSIONS: A previous study demonstrated that c-myc amplification could not be detected in various liposarcomas, but the present unique liposarcoma showed c-myc amplification, so the c-myc amplification may indicate that the present liposarcoma is an LFS-related tumor. The present case further clarifies the pathological features of MPLPS and LFS-related liposarcomas by broadening their histopathological and genetic diversities.

Novel POLE mutations identified in patients with IMAGE-I syndrome cause aberrant subcellular localisation and protein degradation in the nucleus.

BACKGROUND: DNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding DNA polymerase epsilon catalytic subunit 1 (POLE). However, the underlying molecular mechanisms remain largely unresolved. METHODS: The clinical manifestations in two patients with IMAGE-I syndrome were characterised. Whole-exome sequencing was performed and altered mRNA splicing and protein levels of POLE were determined. Subcellular localisation, cell cycle analysis and DNA replication stress were assessed using fibroblasts and peripheral blood from the patients and transfected cell lines to determine the functional significance of POLE mutations. RESULTS: Both patients presented with growth retardation, adrenal insufficiency, immunodeficiency and complicated diffuse large B-cell lymphoma. We identified three novel POLE mutations: namely, a deep intronic mutation, c.1226+234G>A, common in both patients, and missense (c.2593T>G) and in-frame deletion (c.711_713del) mutations in each patient. The unique deep intronic mutation produced aberrantly spliced mRNAs. All mutants showed significantly reduced, but not null, protein levels. Notably, the mutants showed severely diminished nuclear localisation, which was rescued by proteasome inhibitor treatment. Functional analysis revealed impairment of cell cycle progression and increase in the expression of phospho-H2A histone family member X in both patients. CONCLUSION: These findings provide new insights regarding the mechanism via which POLE mutants are highly susceptible to proteasome-dependent degradation in the nucleus, resulting in impaired DNA replication and cell cycle progression, a characteristic of DNA replisome-associated diseases.

A novel 8.57-kb deletion of the upstream region of PRKAR1A in a family with Carney complex.

Carney complex (CNC) is a rare hereditary syndrome that involves endocrine dysfunction and the development of various types of tumors. Chromosome 2p16 and PRKAR1A on chromosome 17 are known susceptibility loci for CNC. Here we report a mother and son with CNC caused by an 8.57-kb deletion involving the transcription start site and non-coding exon 1 of PRKAR1A. The proband is a 28-year-old male with bilateral large-cell calcified Sertoli cell testicular tumors and pituitary adenoma. Comprehensive genomic profiling for cancer mutations using Foundation One CDx failed to detect any mutations in PRKAR1A in DNA from the testicular tumor. Single-nucleotide polymorphism array analysis of the proband's genomic DNA revealed a large deletion in the 5' region of PRKAR1A. Genomic walking further delineated the region an 8.57-kb deletion. A 1.68-kb DNA fragment encompassed by the deleted region showed strong promoter activity in a NanoLuc luciferase reporter assay. The patient's mother, who is suffering from recurrent cardiac myxoma, a critical sign for CNC, carried an identical deletion. The 8.57-kb deleted region is a novel lesion for CNC and will facilitate molecular diagnosis of the disease.

Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated ß-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and ß-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of ß-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the ß-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

Duplications in the G3 domain or switch II region in HRAS identified in patients with Costello syndrome.

Costello syndrome (CS) is an autosomal-dominant disorder characterized by distinctive facial features, hypertrophic cardiomyopathy, skeletal abnormalities, intellectual disability, and predisposition to cancers. Germline variants in HRAS have been identified in patients with CS. Intragenic HRAS duplications have been reported in three patients with a milder phenotype of CS. In this study, we identified two known HRAS variants, p.(Glu63_Asp69dup), p.(Glu62_Arg68dup), and one novel HRAS variant, p.(Ile55_Asp57dup), in patients with CS, including a patient with craniosynostosis. These intragenic duplications are located in the G3 domain and the switch II region. Cells expressing cDNA with these three intragenic duplications showed an increase in ELK-1 transactivation. Injection of wild-type or mutant HRAS mRNAs with intragenic duplications in zebrafish embryos showed significant elongation of the yolk at 11 h postfertilization, which was improved by MEK inhibitor treatment, and a variety of developmental abnormalities at 3 days post fertilization was observed. These results indicate that small in-frame duplications affecting the G3 domain and switch II region of HRAS increase the activation of the ERK pathway, resulting in developmental abnormalities in zebrafish or patients with CS.

Detection of intracellular histological abnormalities using cardiac magnetic resonance T1 mapping in patients with Danon disease: a case series.

BACKGROUND: Danon disease is an X-linked dominant disorder with defects in the lysosome-associated membrane protein 2 (LAMP2) gene and is characterized histologically by intracellular autophagic vacuoles in skeletal and cardiac muscles. Cardiac magnetic resonance (CMR) T1 mapping potentially allows to differentiate intracellular and extracellular cardiac abnormalities with a combination of native T1 value and extracellular volume (ECV) fraction. CASE SUMMARY: We assessed CMR T1 mapping in two Danon disease patients (a 22-year-old man and his 48-year-old mother), who had a LAMP2 c.864G>A p. Val288Val mutation, and two blood relatives without Danon disease (his 47-year-old maternal aunt and 49-year-old father). The male patient underwent a left ventricular (LV) assist device implantation at 15 months after the image acquisition because he was inotrope dependent (INTERMACS profile 3) and had no noticeable psychological or musculoskeletal symptoms. His mother was in New York Heart Association Class II with mildly reduced LV ejection fraction (46%). The Danon group showed late gadolinium enhancement (LGE) in the anterior and posterolateral LV walls. In the interventricular wall, where evident LGE was not noted, the Danon group had high native T1 value, compared with the T1 value in the non-Danon group, and normal ECV fraction. Cardiac biopsy from the interventricular wall showed intracytoplasmic autophagic vacuoles, which are characteristics of Danon disease. DISCUSSION: This characteristic pattern of high native T1 and normal ECV fraction in the areas without LGE, which may reflect the existence of intracytoplasmic autophagic vacuoles, may support the differential diagnosis of Danon disease from other cardiomyopathies.

A novel deletion in the C-terminal region of HSPB8 in a family with rimmed vacuolar myopathy.

Heat shock protein family B member 8, encoded by HSPB8, is an essential component of the chaperone-assisted selective autophagy complex, which maintains muscle function by degrading damaged proteins in the cells. Mutations in HSPB8 have been reported to cause Charcot-Marie-Tooth type 2L, distal hereditary motor neuropathy IIa, and rimmed vacuolar myopathies (RVM). In this study, we identified a novel heterozygous frameshift variant c.525_529del in HSPB8 in a large Japanese family with RVM, using whole exome sequencing. Three affected individuals had severe respiratory failure, which has not been addressed by previous studies. Muscle atrophy in the paraspinal muscles was also a clinical feature of the individuals affected with RVM in this study. The frameshift mutation was located in the last coding exon, and the mutated protein was predicted to harbor an isoleucine-leucine-valine (ILV) sequence, which corresponds to the IXI/V (isoleucine, X amino acids, and isoleucine or valine) motif. The IXI/V motif is essential for assembly into larger oligomers in other small heat shock proteins and all frameshift mutants of HSPB8 were predicted to share the ILV sequence in the C-terminal extension. The in silico prediction tools showed low protein solubility and increased aggregation propensity for the region around the ILV sequence. The IXI/V motif might be associated with the pathogenesis of HSPB8-related RVM.