PHOX2B mutations and genetic predisposition to neuroblastoma.

Germline Mutations of the Paired–Like Homeobox 2B ( PHOX2B) Gene in Neuroblastoma

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

To investigate the diagnostic utility of immunohistochemistry for paired-like homeobox 2B (PHOX2B) expression in neuroblastomas (NBs) and tumors that mimic them, tissue samples (n = 229) from 157 cases of NB, 210 central nervous system tumors, and 170 extracranial non-NB solid tumors (n = 170) were immunostained for PHOX2B. Additionally, PHOX2B expression in67 body fluid cytology specimens was analyzed. In tissue specimens, PHOX2B expression was positive in NBs, pheochromocytomas, and paragangliomas but negative in all of the other tumors evaluated. PHOX2B was detected by immunohistochemistry in 5 NB cytology specimens; all of the others were negative. These results suggest that PHOX2B may be a sensitive and specific immunohistochemical marker for the pathological diagnosis and differential diagnosis of NB in both tissue and cytology specimens.

Heterozygous mutations in the Paired like homeobox 2b (PHOX2B) gene are causative of congenital central hypoventilation syndrome (CCHS), a rare monogenic disorder belonging to the family of neurocristopathies and due to a defective development of the autonomic nervous system. Most patients manifest sudden symptoms within 1 year of birth, mainly represented by central apnea and cyanosis episodes. The sudden appearance of hypoxic manifestations in CCHS and their occurrence during sleep resemble two other unexplained perinatal disorders, apparent life-threatening event (ALTE) and sudden and unexpected infant death (SUID), among which the vast majority is represented by sudden infant death syndrome (SIDS). Differently from CCHS, characterized by Mendelian autosomal dominant inheritance, ALTE and SIDS are complex traits, where common genetic variants, together with external factors, may exert an additive effect with symptoms likely manifesting only over a “threshold.” Given the similarities observed among the three abovementioned perinatal disorders, in this work, we have analyzed the frequency of PHOX2B common variants in two groups of Italian idiopathic ALTE (IALTE) and SUIDs/SIDS patients. Here, we report that the c*161G>A (rs114290493) SNP of the 3′UTR PHOX2B (i) became overrepresented in the two sets of patients compared to population matched healthy controls, and (ii) associated with decreased PHOX2B gene expression, likely mediated by miR-204, a microRNA already known to bind the 3′UTR of the PHOX2B gene. Overall, these results suggest that, at least in the Italian population, the SNP c*161G>A (rs114290493) does contribute, presumably in association with others mutations or polymorphisms, to confer susceptibility to sudden unexplained perinatal life-threatening or fatal disorders by increasing the effect of miR-204 in inducing PHOX2B expression down-regulation. However, these are preliminary observations that need to be confirmed on larger cohorts to achieve a clinical relevance.

Congenital central hypoventilation syndrome (CCHS) is a rare disorder affecting the autonomic nervous system that is caused by variants in the paired-like homeobox 2B (PHOX2B) gene. About 10% of patients with CCHS have nonpolyalanine repeat mutations (NPARM) that are associated with severe phenotypes requiring continuous assisted ventilation, Hirschsprung's disease, and increased neural crest tumor risk. However, some patients with NPARM have milder phenotypes. Our objective was to describe the phenotypes in patients with CCHS PHOX2B NPARM. Retrospective case series of patients with CCHS PHOX2B NPARM was conducted at 2 children's hospitals to evaluate their phenotypes. We identified 8 patients with CCHS PHOX2B NPARM aged 3-31 years. Seven patients were diagnosed in infancy and 1 patient at 2 years of age. All patients presented with respiratory depression in the first 2 months of life. Only 1 patient was identified with a severe phenotype requiring continuous assisted ventilation, Hirschsprung's disease, and a neural crest tumor, which was resected. Five patients required positive pressure ventilation via tracheostomy only during sleep and 2 patients required oxygen only during sleep. Four patients had Hirschsprung's disease and 1 patient had a cardiac pacemaker due to a bradyarrhythmia. None of the patients had echocardiographic abnormalities. Patients with CCHS PHOX2B NPARM can have variable phenotypes, emphasizing the importance of implementing a plan of care that is individualized for each patient. The type of NPARM and their respective location on the PHOX2B gene may play a critical role in the severity of phenotypes displayed by each patient. Kasi AS, Li H, Jurgensen TJ, Guglani L, Keens TG, Perez IA. Variable phenotypes in congenital central hypoventilation syndrome with PHOX2B nonpolyalanine repeat mutations. J Clin Sleep Med. 2021;17(10):2049-2055.

Heterozygous 24-polyalanine repeats in the <i>PHOX2B</i> gene with different manifestations across three generations

Generation and characterization of iPSC lines (BGUi004-A, BGUi005-A) from two identical twins with polyalanine expansion in the paired-like homeobox 2B (PHOX2B) gene

Pathogenic variants in Paired-Like Homeobox 2B (PHOX2B) gene cause congenital central hypoventilation syndrome (CCHS), a rare disorder of the nervous system characterized by absent or reduced ventilatory response to hypoxia and hypercapnia. The focus of management in CCHS is optimizing ventilation. Thus far, no medication has proved effective in improving ventilation. Most CCHS cases are caused by polyalanine repeat expansion mutations. Non-polyalanine repeat expansion mutations are the cause in 8% of cases and result in a more severe clinical presentation. PHOX2B has 3 exons. Exon 3 of PHOX2B is the most common location for CCHS-causing mutations. Thus far, only 9 CCHS-causing mutations have been reported in exon 1, 8 of which were nonsense mutations. We report a child with CCHS who was found to have a novel heterozygous missense variant in exon 1; c.95A > T. Improvement in his apneic episodes was observed following treatment with carbamazepine.

Congenital Central Hypoventilation Syndrome in Children

Introduction: Neuroblastoma (NBL) is the most common type of extracranial pediatric solid tumor. NBL exhibits a wide spectrum of phenotypes, from low-risk cases that spontaneously regress to high-risk cases with 50% mortality, and it is responsible for 12% to 15% of deaths due to childhood cancer. While genome-wide association studies have identified a handful of risk loci, the germline genetic origins of sporadic NBL remain largely unknown. Notably, NBL is one of several childhood cancers that tend to co-occur with specific birth defects, which could point to novel genetic origins. For example, the risk of neuroblastoma is increased 3- to 7-fold among children with certain congenital heart defects (CHD). It is hypothesized that this co-occurrence arises from pleiotropic defects in the neural crest cells that are progenitors of both NBL and some heart structures. Based on these lines of evidence, we hypothesize that novel germline risk loci for NBL can be identified through pleiotropic analyses of CHD and NBL cohorts. Therefore, in this study we seek to identify germline risk loci for neuroblastoma by jointly analyzing CHD and NBL data sets with a primary focus on de novo variants. Methods: Using whole genome sequencing data from the Gabriella Miller Kids First Data Resource Center, we will identify NBL and CHD associated loci by testing for high rates of de novo variants (DNV) in each cohort separately and in the two cohorts combined. The testing strategy compares the rate of DNV to the expected locus-specific mutation rate. In our primary analyses, we will focus on genes known to be associated with CHD in order to reduce the multiple testing penalty for that family of loci. A broader analysis will explore coding and regulatory loci genome wide to identify loci associated jointly with NBL and CHD. Results: We acquired whole genome sequencing data of parent-offspring trios for NBL (n=609) and CHD (n=711) from the Gabriella Miller Kids First Data Resource Center. The probands in the NBL cohort was 54% male and 81% Non-Hispanic White, with a median age at diagnosis of 16 months. The probands in the CHD cohort was 59% male and 60% Non-Hispanic White. The most common CHD phenotypes were ventricular septal defect (46%), tetralogy of Fallot (32%), hypoplastic left heart syndrome (28%), atrial septal defect (25%), and right aortic arch (25%). Analyses to identify DNV are ongoing. Conclusion: The identification of germline risk loci associated with NBL will help to inform the developmental origins of this important childhood cancer and may inform novel risk stratification protocols and therapies. This study also aims to help explain the NBL-CHD co-occurrence, and that could shed light on the development of other neural crest cell derived cancers. Citation Format: Alexander Renwick, Yao Yu, Chad Huff, Philip J. Lupo. De novo variants associated with neuroblastoma and congenital heart defects: evidence of pleiotropic effect from 1311 WGS trios [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2506.

Neuroblastoma (NB) is a pediatric solid tumor originating from neural crest cells (NCCs), which are precursors of the sympathetic nervous system. MYCN amplification is a key factor contributing to the poor prognosis of NB. Anaplastic lymphoma kinase (ALK) alterations, including mutations and amplification, activate oncogenic signaling pathways that, together with MYCN amplification, further enhance tumor malignancy. NCCs are mainly classified into cranial NCCs (cNCCs) and trunk NCCs (tNCCs). Recent studies have reported NB development from tNCCs. However, the potential for NB development from cNCCs remains unexplored. In this study, we sought to mimic the tumorigenic process of NB by overexpressing MYCN and ALK in cNCCs derived from human induced pluripotent stem cells. These modified cells, when subcutaneously transplanted into immunodeficient mice, induced NB-like tumors and could thus be used as an invitro model to study this tumor. Through extensive gene expression profiling and whole-exome sequencing of MYCN/ALK-induced clones, we identified key features of NB, including loss of NF1 and gain of 17q chromosome, which are critical for the development of malignant tumor. This model provides a valuable platform for studying the biological mechanisms driving ALK and MYCN amplification in NB derived from cNCCs.

Neuroblastoma is a childhood malignant form of cancer that originates from neuroblasts, and affects children under five years of age and rarely occurs in adults. It is a tumor that arises from neural crest cells and mainly affects ssthe adrenal medulla. There is remarkable genetic heterogeneity and chromosomal abnormality that leads to this malignant pediatric tumor. Purpose: This article is mostly focused on genetic and molecular abnormalities that lead to neuroblastoma. Heterogeneity of MYCN gene and over-expression of MYCN gene as well as translocations or mutation in gene like ALK (anaplastic lymphoma kinase), PHOX2b gene, rearrangements of ATRX and TERT and also chromosomal deletions and gain that are associated with neuroblastoma that are also discussed. Moreover, in this article associated target therapy against specific genes is also discussed.

The prognosis of high-risk and relapsed neuroblastoma (NB) patients remains poor. The identification of tumor-associated markers is important for differential diagnosis, prognosis, and the development of targeted therapies. The aim of the study was to determine the expression profile of nine most common NB antigens and assess their association with clinicopathological characteristics and patient survival. Tumor samples from 86 patients with NB were evaluated for the expression of tumor-associated antigen (TAA) using quantitative PCR. Twenty-one patients with benign tumors and 17 healthy donors were assigned as controls. Overexpression of tyrosine hydroxylase (TH), PHOX2B, PRAME, GPC2, B7-H3, and Survivin is the most typical for NB. Positive expression of MAGEA3, MAGEA1, and NY-ESO-1 at low levels was detected in 54%, 48%, and 52%, respectively, and was not NB specific. Higher TH expression was observed in samples without MYCN-amplification, while higher expression of Survivin, PHOX2B, and GPC2 was significantly associated with the presence of 1p.36 deletion. Overexpression of TH, PHOX2B, and MAGEA1 was associated with better event-free (EFS) and overall survival (OS). Survivin overexpression was associated with poor EFS but had no impact on OS. Multivariate analysis confirmed Survivin as independent marker for poor survival, and PHOX2B and MAGEA1 for better survival. High expression of TH, PHOX2B, and MAGEA1 genes are favorable prognostic factors for OS and EFS, whereas high expression of Survivin is associated with an increased risk of relapse or progression.

Congenital central hypoventilation syndrome (CCHS) is a rare genetic disorder affecting respiratory control and autonomic nervous system function caused by variants in the paired-like homeobox 2B (PHOX2B) gene. Although most patients are diagnosed in the newborn period, an increasing number of patients are presenting later in childhood, adolescence, and adulthood. Despite hypoxemia and hypercapnia, patients do not manifest clinical features of respiratory distress during sleep and wakefulness. CCHS is a lifelong disorder. Patients require assisted ventilation throughout their life delivered by positive pressure ventilation via tracheostomy, noninvasive positive pressure ventilation, and/or diaphragm pacing. At different ages, patients may prefer to change their modality of assisted ventilation. This requires an individualized and coordinated multidisciplinary approach. Additional clinical features of CCHS that may present at different ages and require periodic evaluations or interventions include Hirschsprung’s disease, gastrointestinal dysmotility, neural crest tumors, cardiac arrhythmias, and neurodevelopmental delays. Despite an established PHOX2B genotype and phenotype correlation, patients have variable and heterogeneous clinical manifestations requiring the formulation of an individualized plan of care based on collaboration between the pulmonologist, otolaryngologist, cardiologist, anesthesiologist, gastroenterologist, sleep medicine physician, geneticist, surgeon, oncologist, and respiratory therapist. A comprehensive multidisciplinary approach may optimize care and improve patient outcomes. With advances in CCHS management strategies, there is prolongation of survival necessitating high-quality multidisciplinary care for adults with CCHS.

Neuroblastoma is a pediatric tumor that is thought to arise from autonomic precursors in the neural crest. Mutations in the PHOX2B gene have been observed in familial and sporadic forms of neuroblastoma and represent the first defined genetic predisposition for neuroblastoma. Here, we address the mechanisms that may underlie this predisposition, comparing the function of wild-type and mutant Phox2b proteins ectopically expressed in proliferating, embryonic sympathetic neurons. Phox2b displays a strong antiproliferative effect, which is lost in all Phox2b neuroblastoma variants analyzed. In contrast, an increase in sympathetic neuron proliferation is elicited by Phox2b variants with mutations in the homeodomain when endogenous Phox2b levels are lowered by siRNA-mediated knockdown to mimic the situation of heterozygous PHOX2B mutations in neuroblastoma. The increased proliferation is blocked by Hand2 knockdown and the antiproliferative Phox2b effects are rescued by Hand2 overexpression, implying Hand2 in Phox2b-mediated proliferation control. A Phox2b variant with a nonsense mutation in the homeodomain elicits, in addition, a decreased expression of characteristic marker genes. Together, these results suggest that PHOX2B mutations predispose to neuroblastoma by increasing proliferation and promoting dedifferentiation of cells in the sympathoadrenergic lineage.