Gordon Holmes syndrome and Huntington-like disease: Two types of RNF216-related disorders

Background and purpose: Cases of RNF216-related disorders have been reported sporadically. The systemic clinical classi�cation and phenotype-genotype correlations of these disorders have not been fully studied. Methods: We report the case of a patient with a novel homozygous mutation in RNF216 and review the cases of all patients with RNF216 mutations reported in the literature. These patients were classi�ed by clinical phenotypes into those with Gordon Holmes syndrome (GHS) and those with Huntington-like disease (HLD). Clinical and genetic features were compared between the groups. Results: The cases of twenty-one patients from 14 families with RNF216 mutations were identi�ed and collected. Eleven (52.4%) patients presented with GHS, and eight (38.1%) presented with HLD. GHS patients had a younger age of onset (24.2 vs. 36.9 years, P<0.001) and a higher percentage of male patients (81.8% vs. 25.0%, P=0.024) than HLD patients. Five (62.5%) HLD patients had ataxia, but only one (9.1%) GHS patient developed chorea. Male patients had a higher percentage of poor pubertal development than female patients (69.2% vs. 12.5%, P=0.024). White matter lesions and cerebellar atrophy were the most common imaging �ndings in both groups. The mutations in nine (64.3%) families were inherited in a monogenic recessive pattern, whereas the mutations in �ve (35.7%) were inherited in an oligogenic pattern by acting with mutations in OTUD4, SRA1 or other unknown genes. All eight mutations found in GHS patients and 57.1% (4/7) of mutations found in HLD patients resulted in amino acid changes or the truncation of the “RING-between-RING” (RBR) domain, which plays a key role in the ubiquitin E3 ligase activity of RNF216. Conclusion: Patients with RNF216 mutations mainly presented with GHS or HLD in a monogenic autosomal-recessive pattern or an oligogenic pattern. The mutations in GHS patients affected the RBR domain and are thought to abrogate ubiquitin E3 ligase activity. The pathogenic mechanism underlying RNF216-related HLD is still unknown


Introduction
The gene RNF216 is an E3 ubiquitin ligase of the "RING-between-RING" (RBR) class that attaches ubiquitin to protein substrates, designating them for proteasome-mediated degradation or signal transduction.(1)Gordon Holmes syndrome (GHS) is an autosomal-recessive cerebellar ataxia characterized by ataxia, dementia and hypogonadotropic hypogonadism.In 2013, David et al reported RNF216 mutations in patients with GHS for the rst time.(2)Subsequently, more studies on RNF216related disorders have been published.(3)(4)(5)(6)(7)(8)(9)(10) In addition to GHS, some of those patients presented with Huntington-like disease (HLD), 4H syndrome (hypodontia, hypomyelination, ataxia and hypogonadotropic hypogonadism) and congenital hypogonadotropic hypogonadism (CHH), con rming the clinical heterogeneity of RNF216-related disorders.(3,4,(8)(9)(10) The inheritance pattern of RNF216-related disorders includes a monogenic recessive pattern and an oligogenic pattern by acting with other genes.(2,4,10) Several different types of mutations have been observed, comprising missense, nonsense, splice site, and frameshift mutations and large deletions.(2,5,9) Although the number of case reports on RNF216-related disorders has been gradually increasing, a comprehensive review that covers all patients thus far is missing.Moreover, group comparisons and phenotype-genotype correlations have not been studied before.
In this study, we report the case of a patient with a novel homozygous mutation in RNF216 and summarize the clinical and genetic features of all described patients.Furthermore, we aim to explore the phenotype-genotype correlations by comparing the clinical and genetic characteristics between different groups.

Patients
We performed whole-exome sequencing (WES) in a patient with the GHS phenotype using the Illumina HiSeq sequencing platform.The variants identi ed in WES were validated by Sanger sequencing.The disease-causing mutations were identi ed by bioinformatics analysis and pedigree analysis.The clinical and imaging information of the patient was collected and analysed.
A systematic literature review was performed via PubMed using the keyword "RNF216" in November 2021.Twenty patients with RNF216 mutations from 13 families were identi ed.The clinical and genetic information of these patients was collected from the literature.

Study design
GHS was de ned as an autosomal-recessive cerebellar ataxia characterized by ataxia, dementia and hypogonadotropic hypogonadism.HLD was de ned as characteristic clinical features of chorea, behavioural disturbances, dementia, and a lack of CAG repeat expansion in the HTT gene.According to the clinical manifestations, all of the patients were classi ed into a GHS group or a HLD group except for two, who were reported to have 4H syndrome or CHH.The clinical and imaging characteristics, as well as the mutation distribution, were compared between the groups.

Data analysis
Student's t test was used for continuous data, and Fisher's exact test (two-tailed) was used for categorical data.P<0.05 was considered statistically signi cant.All statistical analyses were performed using IBM SPSS Statistics version 26.

Case report
A 27-year-old male born to consanguineous Chinese parents (Figure 1A) presented with progressive slurred speech, balance di culties and memory decline since the age of 26.He had poor development of secondary sexual characteristics due to hypogonadotropic hypogonadism (Additional le 1: Table S1).
There was no relevant family history.Neurological examination showed dysarthria, lower limb and gait ataxia, as well as brisk tendon re exes.No nystagmus was noted.The Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores were 30 and 24, respectively.A brain magnetic resonance imaging (MRI) series demonstrated diffuse white matter lesions and cerebellar atrophy years before the onset of neurological symptoms (Figure 1B-M).Fluid attenuated inversion recovery (FLAIR) imaging revealed patchy and con uent white matter lesions affecting the brainstem and the periventricular and subcortical areas.Hyperintensity in the thalami was found on FLAIR images (Figure 1C, 1G, 1K).Neither gadolinium-enhanced lesions nor diffusion-restricted lesions were detected.
The patient was clinically diagnosed with GHS.Whole-exome sequencing identi ed a novel homozygous nonsense mutation c.1549C>T (p.R517X) in the RNF216 gene (NM_ 207111.3).This mutation was predicted to create a premature stop codon, thus producing a truncated protein without a key RBR domain and C-terminal extension (Figure 2).Both parents were unaffected carriers of this mutation.This mutation was classi ed as pathogenic according to the American College of Medical Genetics (ACMG) guidelines.
The mutations of nine (64.3%) families were inherited in monogenic recessive mode, and the mutations of 5 (35.7%) families were inherited in oligogenic mode by acting with mutations in OTUD4 (c.998G>T, NM_001102653.1),SRA1 (c.2374G>A, NM_001035235.3)or other unknown genes.All the mutations in GHS patients and 4 (57.1%)mutations in HLD patients resulted in amino acid changes or the truncation of the RBR domain.Another three mutations (c.1367G>A, c.1616A>G and a deletion of exon 2) in HLD patients were outside the RBR domain.The remaining two mutations in patients with 4H syndrome or CHH were located in the C-terminal extension of the RBR domain.GHS patients tended to have more mutations in uencing RBR domain than HLD patients, although the difference did not reach statistical signi cance (100% vs. 57.1%,P=0.077).

Discussion
this article, we report the case of a Chinese patient with a novel homozygous mutation, c.1549C>T, in RNF216 and review the clinical, neuroimaging and genetic data of all patients with RNF216 mutations published in the literature thus far.
RNF216 disorders span a phenotypic continuum characterized by variable combinations of cerebellar ataxia, chorea, cognitive decline and hypogonadotropic hypogonadism.According to the clinical features, patients with RNF216 mutations were categorized as having GHS or HLD, except for two with 4H syndrome or CHH.Four genes related to GHS include RNF216, either alone or in association with OTUD4, STUB1 and PNPLA6.(2,11,12) RNF216-related GHS is characterized by dementia and con uent white matter lesions, which are rare in GHS patients with other gene mutations.(12,13) In our review, GHS patients with RNF216 mutations developed neurological symptoms in their 20s.Over 90% of those patients typically presented with ataxia, cognitive decline and dysarthria.HLD was another common clinical phenotype of RNF216-related disorders and characterized by chorea, cognitive decline, and psychological and behavioural abnormalities.Interestingly, we found that the average age at the onset of neurological symptoms in HDL patients was approximately 12 years older than that in GHS patients.The HLD group was predominantly female, whereas the GHS group was predominantly male.Despite the clinical heterogeneity, there was also an overlap between different phenotypes.For instance, cognitive decline was very common in both groups.More than half of HLD patients developed ataxia during the course of disease, and one GHS patient had accompanying chorea.Apart from GHS and HLD, a male patient with the homozygous mutation c.2453-2A>G in RNF216 was reported to present with 4H syndrome.(3)However, it is still controversial to classify the patient as having 4H syndrome on the basis of atypical dental abnormalities and imaging manifestations.Nicole et al considered that this patient should be diagnosed with GHS rather than 4H syndrome.(14) Furthermore, a 19-year-old male patient, in whom digenic mutations in RNF216 (c.2374G>A) and SRA1 (c.2374G>A, NM_001035235.3)were observed, presented with CHH without obvious neurological symptoms or MRI abnormalities.(10) Considering the age at the onset of neurological symptoms in other patients with RNF216 mutations, we thought this patient might be too young to show neurological symptoms.
Another prominent feature in patients with RNF216 mutations is hypogonadotropic hypogonadism due to diminished GnRH secretion and pituitary dysfunction.(2)Interestingly, male patients seemed to be more vulnerable than female patients.Most male patients suffered from poor pubertal development.In contrast, the majority of female patients had normal puberty, and three of them delivered successfully.(4,8) This phenomenon was consistent with the results of previous animal experiments.The targeted deletion of the RNF216 gene in mice resulted in disruption in spermatogenesis and male infertility, but RNF216 was not required for female fertility.(15) White matter lesions and cerebellar atrophy were the most common imaging ndings and were present in over 90% of patients.Cerebral atrophy and the involvement of the brainstem, basal ganglia and thalami were also observed in some patients.However, gadolinium-enhanced lesions or diffusion-restricted lesions were not detected in any patient.Characteristic bilateral white matter lesions surrounding the basal ganglia, which have been reported to be related to Huntington's disease,(16) were exclusively found in patients with chorea.(4,6,9) RNF216 is a ubiquitin ligase of the RBR class that attaches ubiquitin chains to substrates.For RNF216, various substrates have been reported, including TOLL-like receptors, tumour necrosis factor-receptor associated factor 3 (TRAF3), the autophagy regulator Beclin and the synaptic regulator activity regulated cytoskeleton associated protein (ARC).(1,(17)(18)(19) The ubiquitin ligase activity of RNF216 requires not only the RBR domain but also an additional C-terminal extension of the RBR domain.(19,20) All mutations in patients with GHS, 4H or CHH resulted in amino acid changes or the truncation of the RBR domain/C-terminal extension of the RBR domain, which was presumed to abrogate ubiquitin E3 ligase activity.However, three of seven mutations (c.1367G>A, c.1616A>G and a deletion in exon 2) in HLD patients did not affect the RBR region/C-terminal extension of the RBR domain.Further functional studies con rmed that the two missense mutations (c.1367G>A and c.1616A>G) in HLD patients reversed the E3 ubiquitin ligase activity of RNF216, implying that these mutations caused disease by mechanisms other than by in uencing ubiquitin E3 ligase activity.(19) These ndings indicated that RNF216-related GHS was strongly associated with mutations affecting the ubiquitin E3 ligase activity of RNF216.However, the pathogenic mechanism underlying RNF216-related HLD is more complicated and remains to be explored in future studies.
In summary, patients with RNF216-related disorders typically presented with GHS or HLD in a monogenic autosomal-recessive pattern or an oligogenic pattern.Mutations in GHS patients were closely related to the RBR domain and ubiquitin E3 ligase activity of RNF216.However, the pathogenic mechanism underlying RNF216-related HLD remains to be explored in future studies.

Abbreviations
Holmes syndrome GHS Huntington-like disorders HLD hypodontia, hypomyelination, ataxia and hypogonadotropic hypogonadism 4H syndrome congenital hypogonadotropic hypogonadism CHH RING-between-RING RBR Declarations Ethics approval and consent to participate The institutional ethics committee of Beijing Tian Tan Hospital, Capital Medical University approved this study.Written, informed consent was obtained from the patient.

Consent for publication
Consent to publish has been obtained from participants to report individual patient data.

Figures Figure 1
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Table 1
Summarized clinical and imaging features of RNF216-related GHS and HLD