ROHHAD + ZSCAN1 Publications
FAQ
What is ZSCAN1 and why is it relevant to ROHHAD?
The Zinc finger and SCAN domain-containing protein 1 (ZSCAN1) is an intracellular protein identified as a molecular antigen targeted by autoantibodies in patients with ROHHAD syndrome. ZSCAN1 is presumed to have an intracellular localization (inside the cell). It is expressed in the neuroblastic tumor (NT) tissue associated with ROHHAD. It is also expressed in healthy human hypothalamus tissue, which is the target tissue affected in ROHHAD patients.
ZSCAN1 autoantibodies (ZSCAN1-abs) were first identified in ROHHAD patients with neuroblastic tumors, where they were present in over 70% of cases and proposed as a biomarker for paraneoplastic neurological syndrome. However, subsequent studies have shown that these antibodies are also present in the majority of patients without tumors, with positivity rates exceeding 80% in both serum and CSF samples. This indicates that ZSCAN1-abs are not dependent on tumor presence and may instead reflect a broader immune-mediated mechanism underlying ROHHAD. Importantly, ZSCAN1-abs are not universally detected: some patients with tumors test negative, while others without tumors test positive. Thus, while not an absolute marker, ZSCAN1-abs remain a strong diagnostic candidate for ROHHAD across both tumor-associated and idiopathic forms of the syndrome.
Is the ZSCAN1 marker only found in ROHHAD individuals with neural crest tumors?
What methods are used to test for ZSCAN1 autoantibodies?
The identification and diagnostic testing of ZSCAN1 autoantibodies (ZSCAN1-abs) in patients with ROHHAD syndrome rely on several complementary molecular and cell-based methods. The initial discovery came from Phage-Display Immunoprecipitation and Next-Generation Sequencing (PhIP-Seq), which screened cerebrospinal fluid (CSF) from affected children. This approach identified ZSCAN1 as the only consistently enriched protein, establishing it as the lead candidate autoantigen.
For broader application and clinical screening, a customized ELISA was developed, allowing quantitative measurement of anti-ZSCAN1 antibodies. This assay proved particularly valuable in patients without tumor association, with over 85% testing positive. In parallel, comprehensive protein array analysis enabled wide-scale screening of thousands of proteins and reaffirmed ZSCAN1 as the relevant autoantigen in ROHHAD, even in idiopathic cases. Together, these methods have established ZSCAN1-abs as a reproducible and clinically meaningful biomarker for ROHHAD.
Why is the importance of ZSCAN1 testing to the ROHHAD population?
The discovery of ZSCAN1 autoantibodies (ZSCAN1-abs) provides the first molecular biomarker for ROHHAD syndrome. Their presence suggests an immune-mediated etiology, linking ROHHAD to both hypothalamic dysfunction and neuroblastic tumors, though antibodies can also be detected in patients without tumors. Testing, particularly in CSF samples, allows for earlier and more accurate diagnosis than relying on clinical criteria alone. Clinically, ZSCAN1-abs support the use of immunotherapy, guide tumor screening, and may help predict prognosis. Importantly, this finding emphasizes the value of human-specific research methods, as ZSCAN1 has no rodent equivalent and was identified through PhIP-Seq.
Published ARticles
“ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD” by mandel-brehm et al. (2022)
This article describes the discovery of autoantibodies targeting the protein ZSCAN1 as a robust molecular biomarker associated with Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD) syndrome, providing strong evidence that the disease is a novel pediatric Paraneoplastic Neurological Syndrome (PNS).
Due to the failure of classical methods (like immunohistochemical screening against rodent brain tissue) to identify anti-neural reactivity in ROHHAD patient CSF, researchers deployed a human-specific approach:
Discovery (PhIP-Seq): Immunoglobulin G (IgG) from pediatric ROHHAD patients (n=9) and controls (n=125 total) was screened. Of approximately 20,000 protein possibilities screened, only one protein, ZSCAN1, satisfied the strict criteria for enrichment in the ROHHAD cohort.
Orthogonal Validation: The association of ZSCAN1 was reproduced and confirmed in 7 out of 9 ROHHAD patients using two orthogonal assays.
Findings:
Tumor Association: All seven ROHHAD patients (ROHHAD-1 through -7) who tested positive for ZSCAN1 autoantibodies had NTs identified.
Tissue Expression: ZSCAN1 expression was confirmed in both the ROHHAD-associated tumor tissue (via immunohistochemistry) and in healthy human hypothalamus tissue (via mass spectrometry), fulfilling the requirement of an onconeural antigen.
Implications for Testing and Diagnosis: Diagnostic Biomarker: Autoantibodies to ZSCAN1 are a putative biomarker of ROHHAD, offering a means toward earlier, accurate diagnosis of this severe disorder.
The findings provide a robust finding of autoantibodies to ZSCAN1 as a marker for tumor-associated pediatric ROHHAD, and collectively support the suspicion that ROHHAD is a novel type of PNS. This is the first identified intracellular antigen in a PNS unique to children. The detection of ZSCAN1-abs should aid in diagnosis, highlighting the need for tumor identification, and advancing the use of immunosuppressive treatments, although the response to such therapies can be variable, consistent with PNS involving intracellular antigens.
Full article: ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD-Web of Science Core Collection
“Anti-ZSCAN1 Autoantibodies Are a Feasible Diagnostic Marker for ROHHAD Syndrome Not Associated with a Tumor” by Nakamura-Utsunomiya et al. (2024)
The study retrospectively identified 13 patients who fulfilled the diagnostic criteria for ROHHAD syndrome but without tumor association. Paired serum and Cerebrospinal Fluid (CSF) samples were available for 6 patients, while 7 others had only serum available. The samples were tested for ZSCAN1-abs using an in-house cell-based assay (CBA) utilizing HEK293 cells transfected with human ZSCAN1 cDNA. A large control group of 199 patients with inflammatory or non-inflammatory diseases and healthy participants were also tested.
Key Results:
Antibody Prevalence: ZSCAN1-abs were identified in 5 of 6 patients (83.3%) who had paired serum and CSF samples available.
Sample Preference: Among the positive patients, ZSCAN1-abs were detected only in the CSF in four cases, and in both serum and CSF in one case. This finding, combined with previous reports, suggests that CSF is the sample of choice when ZSCAN1-abs are tested by CBA in patients with suspected ROHHAD syndrome.
Serum Limitations: The remaining seven ROHHAD patients for whom only serum was available tested negative for ZSCAN1-abs.
Control Cohort: Samples from all 199 controls tested were negative for ZSCAN1-abs.
Treatment: Three ZSCAN1-abs positive patients received first-line immunotherapy (such as IV immunoglobulins), and one showed mild improvement after receiving cyclophosphamide.
Conclusions and Implications: The study confirms the previously reported association between ROHHAD syndrome and ZSCAN1-abs in children and expands the clinical profile to patients without an underlying tumor. The presence of ZSCAN1-abs does not necessarily predict the presence of a tumor, indicating that patients with idiopathic ROHHAD syndrome should also be tested for these antibodies. The strong evidence for an immune-mediated pathogenesis, coupled with the disease's poor prognosis, supports a trial of immunotherapy in patients who have severe, progressive, or refractory symptoms. The detection of ZSCAN1-abs in an adult patient suggests that the syndrome may be under-recognized in adults presenting with related symptoms, such as obesity, endocrinological alterations, apneas, and hypoventilation.
Full article: Anti-ZSCAN1 Autoantibodies Are a Feasible Diagnostic Marker for ROHHAD Syndrome Not Associated with a Tumor-Web of Science Core Collection
"Antibodies Against ZSCAN1 in Pediatric and Adult Patients With Non-Paraneoplastic ROHHAD Syndrome” by serafim et al. (2024)
This article investigates the relationship between autoantibodies targeting the zinc finger and SCAN domain-containing protein 1 (ZSCAN1) and ROHHAD syndrome in patients who do not have associated neuroendocrine tumors (NETs).
The researchers used a combination of techniques: They first analyzed a mixture of serum samples from patients with ROHHAD syndrome not associated with an NET and identified 33 candidate antigens. A customized protein array containing these 33 antigens was then used to analyze serum samples of five individual patients with ROHHAD syndrome (Cases 1–5), including four patients without a tumor and one patient (Case 4) diagnosed with ROHHAD syndrome associated with a retroperitoneal tumor. Immunostaining was conducted on mouse subfornical organs (SFOs) to determine the co-expression of ZSCAN1 and patient IgG reactivity. The SFO is a periventricular organ linked to hypothalamic involvement in ROHHAD. Finally, a quantitative, custom-made ELISA for anti-ZSCAN1 autoantibodies was used to analyze a cohort of 14 patients with ROHHAD syndrome not associated with a tumor, 15 normal subjects, and 5 subjects with other autoimmune disorders.
Results:
ZSCAN1 Identified as Antigen: The custom protein array analysis identified ZSCAN1 as an antigen in four of the five patients with ROHHAD syndrome not associated with a tumor. ZSCAN1 was not identified as an antigen in Case 4, who had a retroperitoneal tumor.
Tissue Co-Expression: Immunostaining of mouse SFOs showed that ZSCAN1 was expressed in the SFO, and the IgG in a patient's serum sample (Case 1) exhibited co-reactivity to ZSCAN1 in the same portion of the SFO, suggesting reactivity to ZSCAN1 at this pathological site.
High Prevalence in Non-Tumor Cohort: The custom ELISA demonstrated that 12 of the 14 patients (85.7%) with ROHHAD syndrome not associated with a tumor were positive for anti-ZSCAN1 autoantibodies . Normal subjects and subjects with other autoimmune disorders were negative for anti-ZSCAN1 autoantibodies.
Prognostic Indicator: Notably, patients with a poor prognosis had higher than average anti-ZSCAN1 autoantibody titers (115 ± 85).
The results suggest that anti-ZSCAN1 autoantibodies are a feasible diagnostic marker for ROHHAD syndrome regardless of the presence of a tumor. This finding is critical because the diagnosis of ROHHAD is primarily based on clinical symptoms, and reliable diagnostic methods other than clinical evaluation are greatly needed to improve the prognosis. The level of anti-ZSCAN1 autoantibodies may also be useful in evaluating disease severity and the need for immunosuppressive treatment in patients with ROHHAD syndrome not associated with a tumor.