Establishment and characterization of a novel MDM2/MYCN-co-amplified neuroblastoma cell line, NBN-SHIM, established from a late recurrent stage MS tumor.

The biological heterogeneity of neuroblastoma underscores the need for an in vitro model of each molecularly defined subgroup to investigate tumorigenesis and develop targeted therapies. We have established a permanently growing cell line from a 12-year-old girl who developed a late recurrent stage MS, MDM2-amplified neuroblastoma arising in the liver and performed histological, molecular, cytogenetic, exome, and telomere analyses of the recurrent tumor and the cell line. On histology, the recurrent tumor was immunoreactive for TP53, CDKN1A, and MDM2. A molecular cytogenetic study of the recurrent tumor revealed the amplification of MDM2 but no amplification of MYCN. The established cell line, NBM-SHIM, showed amplification of both MDM2 and MYCN on double-minute chromosomes. A copy number evaluation based on exome data confirmed the finding for MYCN and MDM2 and further identified high ploidy on CDK4 and GLI2 loci in the recurrent tumor and the cell line. The telomere maintenance mechanism on the cell line is unusual in terms of the low expression of TERT despite MYCN amplification and alternative lengthening of telomeres suggested by positive value for C-circle assay and telomere contents quantitative assay. The cell line is unique because it was established from a MYCN-nonamplified, MDM2-amplified, late-relapsed stage MS neuroblastoma, and MYCN amplification was acquired during cell culture. Therefore, the cell line is a valuable tool for investigating neuroblastoma tumorigenesis and new molecular targeted therapies for disrupted ARF-TP53-MDM2 pathway and amplification of MDM2 and CDK4.

Nanopore long-read sequencing analysis reveals ZIC1 dysregulation caused by a de novo 3q inversion with a breakpoint located 7 kb downstream of ZIC1.

Zic family member 1 (ZIC1), a gene located on chromosome 3q24, encodes a transcription factor with zinc finger domains that is essential for the normal development of the cerebellum. Heterozygous loss-of-function of ZIC1 causes Dandy-Walker malformation, while heterozygous gain-of-function leads to a multiple congenital anomaly syndrome characterized by craniosynostosis, brain abnormalities, facial features, and learning disability. In this study, we present the results of genetic analysis of a male patient with clinically suspected Gomez-Lopez-Hernandez syndrome. The patient displayed multiple congenital abnormalities, including bicoronal craniosynostosis, characteristic facial features, cerebellar malformation with rhombencephalosynapsis, and temporal alopecia, and a de novo inversion of chromosome 3q. Breakpoint analysis using a Nanopore long-read sequencer revealed a breakpoint in the distal centromere of 3q24 located 7 kb downstream of the 3' untranslated region of ZIC1. On the basis of the clinical similarities, we concluded that the abnormalities in this patient were caused by the transcriptional dysregulation of ZIC1. We hypothesize the underlying molecular mechanisms of transcriptional dysregulation of ZIC1 such as the abnormalities in topologically associated domains encompassing ZIC1. This study highlights the usefulness of long-read sequencing in the analysis of de novo balanced chromosomal abnormalities.

Death of children with Down syndrome by gestational age and cause.

BACKGROUND: We often encounter preterm infants with Down syndrome (DS) who die in the neonatal intensive care unit (NICU). In this study, we examined survival until NICU discharge and assessed the developmental prognosis of preterm infants with DS. METHODS: We retrospectively reviewed 416 infants with DS hospitalized during the past 27 years at our NICU. RESULTS: Death occurred in 8/20 (40%) infants at <32 weeks' gestation, 11/23 (48%) at 32-33 weeks, 9/99 (9%) at 34-36 weeks, and 9/274 (3%) at >36 weeks. In total, 84% of infants who died and 25% of those who survived had a non-reassuring fetal status (p < 0.001). Sex, small-for-gestational-age status, and postnatal transport were not associated with death. The main causes of death were bronchopulmonary dysplasia in 4/8 (50%) infants at <32 weeks' gestation, transient abnormal myelopoiesis in 11/20 (55%) and lymphatic dysplasia in 6/20 (30%) at 32-36 weeks, and varied causes at >36 weeks. Among survivors born at <34 weeks' gestation, 6/19 (32%) aged >2 years had moderate or severe cerebral palsy. CONCLUSIONS: These data on the high mortality and morbidity of preterm infants with DS may be useful for patient treatment and parent counseling in NICUs treating critically ill infants. IMPACT: Most infants with Down syndrome born at <34 weeks' gestation are born by cesarean section because of the non-reassuring fetal status. The mortality rate before discharge for infants with Down syndrome born at <34 weeks' gestation was 40%, and 30% of survivors developed moderate or severe cerebral palsy. The risk of death due to bronchopulmonary dysplasia and pulmonary hypertension was high in very preterm infants with Down syndrome despite the absence of chorioamnionitis. Infants with Down syndrome were born 1-2 weeks earlier than unaffected controls.

PHACES-like syndrome with TMEM260 compound heterozygous variants.

PHACES syndrome is a multiple congenital disorder with unknown etiology that is characterized by Posterior fossa anomalies, Hemangioma, Arterial lesions, Cardiac abnormalities/coarctation of the aorta, Eye anomalies, and Sternal cleft. Compound heterozygous or homozygous TMEM260 variants cause structural heart defects and renal anomalies syndrome (SHDRA). We describe a 10-year-old male patient with a PHACES-like syndrome and TMEM260 compound heterozygous variants who demonstrated overlapping phenotypes between the two syndromes. He presented with truncus arteriosus, supraumbilical raphe, ophthalmological abnormality, vertebral abnormality, borderline intellectual disability, and hearing loss. He had normal serum creatinine. In proband exome sequencing, compound heterozygous TMEM260 variants (NM_017799.4 c.1617delG p.(Trp539Cysfs*9)/c.1858C > T p.(Gln620*)) were identified. Twelve patients have been reported with TMEM260-related SHDRA: 10 had truncus arteriosus and 6 had renal failure. One previously reported patient had facial port wine nevus and another patient had supraumbilical raphe, which are the cardinal signs for PHACES syndrome. TMEM260-related SHDRA could share overlapping clinical features with PHACES syndrome. This report expands the phenotypic spectrum of a TMEM260-related disorder.

Genetic and clinical landscape of childhood cerebellar hypoplasia and atrophy.

PURPOSE: Cerebellar hypoplasia and atrophy (CBHA) in children is an extremely heterogeneous group of disorders, but few comprehensive genetic studies have been reported. Comprehensive genetic analysis of CBHA patients may help differentiating atrophy and hypoplasia and potentially improve their prognostic aspects. METHODS: Patients with CBHA in 176 families were genetically examined using exome sequencing. Patients with disease-causing variants were clinically evaluated. RESULTS: Disease-causing variants were identified in 96 of the 176 families (54.5%). After excluding 6 families, 48 patients from 42 families were categorized as having syndromic associations with CBHA, whereas the remaining 51 patients from 48 families had isolated CBHA. In 51 patients, 26 aberrant genes were identified, of which, 20 (76.9%) caused disease in 1 family each. The most prevalent genes were CACNA1A, ITPR1, and KIF1A. Of the 26 aberrant genes, 21 and 1 were functionally annotated to atrophy and hypoplasia, respectively. CBHA+S was more clinically severe than CBHA-S. Notably, ARG1 and FOLR1 variants were identified in 2 families, leading to medical treatments. CONCLUSION: A wide genetic and clinical diversity of CBHA was revealed through exome sequencing in this cohort, which highlights the importance of comprehensive genetic analyses. Furthermore, molecular-based treatment was available for 2 families.

A nationwide survey of Schaaf-Yang syndrome in Japan.

Schaaf-Yang syndrome (SYS) is a congenital disorder characterized by developmental delay, autism spectrum disorder and congenital joint contractures. In this study, a nationwide epidemiological questionnaire-based survey of SYS in the Japanese population was conducted to establish patient numbers, clinical features and genetic information. In the primary survey, we investigated the number of SYS patients. In the secondary survey, we obtained and analyzed detailed clinical and genetic information of SYS patients. This survey collected information on 25 genetically-confirmed patients. The major clinical symptoms included neonatal hypotonia (96% of the patients), poor suck in infancy (82%), developmental delay (100%) and joint contractures (83%). Other main symptoms and findings included characteristic facial features (100%), small hands (92%), eye abnormalities (92%) and short stature (79%). Based on the information collected on activities of daily living, 71% of patients were unable to walk, while 67%, 71%, and 81% of patients required full assistance with eating, toileting and bathing, respectively. Regarding inheritability, the genetic analysis of 21 patients revealed that 14 (67%) carried de novo truncating variants in the melanoma antigen L2 (MAGEL2) gene and seven (33%) had inherited truncating variants from their fathers who were carriers. This survey revealed the clinical and genetic features in Japanese SYS patients. The majority of SYS patients required assistance in many aspects of daily living, and there were a certain number of carriers of the imprinting disorder.

Infantile hepatic hemangioma and hepatic mesenchymal hamartoma in an infant associated with placental mesenchymal dysplasia: a case report.

BACKGROUND: Although infantile hepatic hemangioma and hepatic mesenchymal hamartoma are relatively common in benign pediatric liver tumors, coexistence of the two tumors is rare. Placental mesenchymal dysplasia is also a rare disorder. We report the case of a baby girl born after a pregnancy complicated by placental mesenchymal dysplasia, who developed both infantile hepatic hemangioma and hepatic mesenchymal hamartoma. CASE PRESENTATION: The patient was born at 32 weeks and 5 days of gestation for impending placental abruption, weighing 1450 g. Liver tumors, composed of both hypervascular solid and large cystic lesions, were detected after birth and markedly increased to create abdominal distention within 9 months. Diagnostic imaging suspected the coexistence of infantile hepatic hemangioma and cystic hepatic mesenchymal hamartoma. Following propranolol therapy for infantile hepatic hemangioma and needle puncture of a large cyst, the cystic lesions and adjacent hypervascular lesions were partially resected via laparotomy. Pathological findings confirmed the coexistence of hepatic mesenchymal hamartoma and infantile hepatic hemangioma, which had no association with androgenetic/biparental mosaicism. The postoperative course was uneventful, and the tumor had not regrown after 3 years. CONCLUSIONS: Although the coexistence of infantile hepatic hemangioma and hepatic mesenchymal hamartoma associated with placental mesenchymal dysplasia is extremely rare, the pathological and pathogenetic similarities between these disorders suggest that they could have derived from similar embryologic origins rather than being a mere coincidence. Further follow-up is required, with careful attention to the potential for malignant hepatic mesenchymal hamartoma transformation.

Novel COL2A1 variants in Japanese patients with spondyloepiphyseal dysplasia congenita.

Spondyloepiphyseal dysplasia congenita (SEDC) is a multisystemic skeletal disorder caused by pathogenic variants in COL2A1. Here, we report the genotype-phenotype correlations in five Japanese patients with SEDC based on their clinical and radiological findings. All five patients had novel missense variants resulting in glycine substitutions (G474V, G543E, G567S, G594R, and G1170R). Genetic testing is important for early intervention for the extraskeletal complications of SEDC. Spondyloepiphyseal dysplasia congenita (SEDC) (OMIM#183900) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, flattened vertebral bodies (skeletal abnormalities), and extraskeletal features, including myopia, retinal degeneration with retinal detachment, and cleft palate. SEDC is caused by a heterozygous variant in the collagen II alpha 1 (COL2A1) gene.

De novo heterozygous variants in KIF5B cause kyphomelic dysplasia.

Kyphomelic dysplasia is a heterogeneous group of skeletal dysplasias characterized by severe bowing of the limbs associated with other variable findings, such as narrow thorax and abnormal facies. We searched for the genetic etiology of this disorder. Four individuals diagnosed with kyphomelic dysplasia were enrolled. We performed whole-exome sequencing and evaluated the pathogenicity of the identified variants. All individuals had de novo heterozygous variants in KIF5B encoding kinesin-1 heavy chain: two with c.272A>G:p.(Lys91Arg), one with c.584C>A:p.(Thr195Lys), and the other with c.701G>T:p.(Gly234Val). All variants involved conserved amino acids in or close to the ATPase activity-related motifs in the catalytic motor domain of the KIF5B protein. All individuals had sharp angulation of the femora and humeri, distinctive facial features, and neonatal respiratory distress. Short stature was observed in three individuals. Three developed postnatal osteoporosis with subsequent fractures, two showed brachycephaly, and two were diagnosed with optic atrophy. Our findings suggest that heterozygous KIF5B deleterious variants cause a specific form of kyphomelic dysplasia. Furthermore, alterations in kinesins cause various symptoms known as kinesinopathies, and our findings also extend the phenotypic spectrum of kinesinopathies.

Persistent Hyperplastic Primary Vitreous with Microphthalmia and Coloboma in a Patient with Okur-Chung Neurodevelopmental Syndrome.

Okur-Chung neurodevelopmental syndrome is a rare autosomal dominant disorder caused by pathogenic variants in CSNK2A1, which encodes the alpha 1 catalytic subunit of -casein kinase II. This syndrome is characterized by intellectual disability, developmental delay, and multisystemic -abnormalities including those of the brain, extremities, and skin as well as cardiovascular, gastrointestinal, and immune systems. In this study, we describe a 5-year-old boy with a de novo novel nonsense variant in CSNK2A1, NM_001895.3:c.319C>T (p.Arg107*). He showed bilateral persistent hyperplastic primary vitreous with microphthalmia, lens dysplasia, and coloboma. Ocular manifestations are very rare in this syndrome, and this study expands the spectrum of the clinical presentations of this syndrome.

A patient with Silver-Russell syndrome with multilocus imprinting disturbance, and Schimke immuno-osseous dysplasia unmasked by uniparental isodisomy of chromosome 2.

Silver-Russell syndrome (SRS) is a congenital disorder characterized by prenatal and postnatal growth failure and craniofacial features. Hypomethylation of the H19/IGF2:IG-differential methylated region (H19LOM) is observed in 50% of SRS patients, and 15% of SRS patients with H19LOM had multilocus imprinting disturbance (MLID). Schimke immuno-osseous dysplasia (SIOD), characterized by spondyloepiphyseal dysplasia and nephropathy, is an autosomal recessive disorder caused by mutations in SMARCAL1 on chromosome 2. We report a patient with typical SRS-related features, spondyloepiphyseal dysplasia, and severe nephropathy. Molecular analyses showed H19LOM, paternal uniparental isodisomy of chromosome 2 (iUPD(2)pat), and a paternally inherited homozygous frameshift variant in SMARCAL1. Genome-wide methylation analysis showed MLID in this patient, although it showed no MLID in another patient with SIOD without SRS phenotype. These results suggest that iUPD(2)pat unmasked the recessive mutation in SMARCAL1 and that the SMARCAL1 gene mutation may have no direct effect on the patient's methylation defects.

ATP1A3 variants and slowly progressive cerebellar ataxia without paroxysmal or episodic symptoms in children.

A heterogeneous spectrum of clinical manifestations caused by mutations in ATP1A3 have been previously described. Here we report two cases of infantile-onset cerebellar ataxia, due to two different ATP1A3 variants. Both patients showed slowly progressive cerebellar ataxia without paroxysmal or episodic symptoms. Brain magnetic resonance imaging revealed mild cerebellar cortical atrophy in both patients. Whole exome sequencing revealed a de novo heterozygous variant in ATP1A3 in both patients. One patient had the c.460A>G (p.Met154Val) variant, while the other carried the c.1050C>A (p.Asp350Lys) variant. This phenotype was characterized by a slowly progressive cerebellar ataxia since the infantile period, which has not been previously described in association with ATP1A3 variants or in ATP1A3-related clinical conditions. Our report contributes to extend the phenotypic spectrum of ATP1A3 mutations, showing paediatric slowly progressive cerebellar ataxia with mild cerebellar atrophy alone as an additional clinical presentation of ATP1A3-related neurological disorders.

A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic mutation in these isolated cells.

PURPOSE: Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation. METHODS: Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by "lumen digestion" and irrigating the cystic cavity with trypsin, and maintained in culture. RESULTS: The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases. CONCLUSIONS: We established a novel technique for isolating LM-LECs from LM tissue by "lumen digestion" without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.

A Japanese girl with mild xeroderma pigmentosum group D neurological disease diagnosed using whole-exome sequencing.

We report a Japanese girl with mild xeroderma pigmentosum group D neurological disease. She had short stature, cataracts, intellectual disability, and mild skin symptoms. However, she was not clinically diagnosed. Using whole-exome sequencing, we identified compound heterozygous pathogenic variants in ERCC2. In the future, the patient may develop skin cancer and her neurological symptoms may progress. Early genetic testing is necessary to clarify the cause of symptoms in undiagnosed patients.

Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Attitudes toward and current status of disclosure of secondary findings from next-generation sequencing: a nation-wide survey of clinical genetics professionals in Japan.

The management of secondary findings (SFs), which are beyond the intended purpose of the analysis, from clinical comprehensive genomic analysis using next generation sequencing (NGS) presents challenges. Policy statements regarding their clinical management have been announced in Japan and other countries. In Japan, however, the current status of and attitudes of clinical genetics professionals toward reporting them are unclear. We conducted a questionnaire survey of clinical genetics professionals at two time points (2013 and 2019) to determine the enforcement of the SF management policy in cases of comprehensive genetic analysis of intractable diseases and clinical cancer genome profiling testing. According to the survey findings, 40% and 70% of the respondents stated in the 2013 and 2019 surveys, respectively, that they had an SF policy in the field of intractable diseases, indicating that SF policy awareness in Japan has changed significantly in recent years. Furthermore, a total of 80% of respondents stated that their facility had established a policy for clinical cancer genome profiling testing in the 2019 survey. In both surveys, the policies included the selection criteria for genes to be disclosed and the procedure to return SFs, followed by recommendations and proposals regarding SFs in Japan and other countries. To create a better list of the genes to be disclosed, further examination is needed considering the characteristics of each analysis.

Unique skeletal manifestations in patients with Primrose syndrome.

Primrose syndrome (OMIM 259050) is a rare disorder characterised by macrocephaly with developmental delay, a recognisable facial phenotype, altered glucose metabolism, and other features such as sensorineural hearing loss, short stature, and calcification of the ear cartilage. It is caused by heterozygous variants in ZBTB20, a member of the POK family of transcription repressors. Recently, this gene was shown to have a role in skeletal development through its action on chondrocyte differentiation by repression of SOX9. We describe five unrelated patients with Primrose syndrome and distinct skeletal features including multiple Wormian bones, platybasia, bitemporal bossing, bathrocephaly, slender bones, epiphyseal and spondylar dysplasia. The radiological abnormalities of the skull and the epiphyseal dysplasia were the most consistent findings. This novel constellation of skeletal features expands the phenotypic spectrum of the disorder.