Ataxia, Dementia, and Hypogonadotropism Caused by Disordered Ubiquitination

Abstract

BACKGROUND

The combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive.

METHODS

We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model.

RESULTS

Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis.

CONCLUSIONS

The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease. (Funded by the National Institutes of Health and others.)

In recent years, we have seen great advances in the elucidation of genetic causes of cerebellar ataxia, with newly identified genes regulating a wide spectrum of cellular functions, including intracellular signaling, tau regulation, and mitochondrial function.¹ However, a genetic defect cannot be found in approximately 40% of patients with ataxia,¹ including those in whom ataxia is associated with reproductive endocrine failure, a syndrome first reported by Gordon Holmes in 1908.² Most patients with this syndrome have a hypogonadotropic condition, with defective secretion of gonadotropins by the pituitary gland.^3–12 Strikingly, genes associated with ataxia have little functional overlap with genes associated with hypogonadotropic hypogonadism, which encode proteins involved in the biologic function of the neurons that secrete gonadotropin-releasing hormone (GnRH).¹³

A decade ago, we described a consanguineous family with a syndrome of cerebellar ataxia, dementia, and hypogonadotropic hypogonadism.¹² Here we report the results of whole-exome and targeted sequencing performed to identify mutations that underlie the syndrome in this kindred and in unrelated patients.

METHODS

STUDY PATIENTS

Our study included 12 patients with ataxia and hypogonadotropic hypogonadism from eight families. The pedigrees of the index family and four of the other seven families are shown in Figure 1. The patients were referred to the Massachusetts General Hospital for clinical or genetic evaluation between 2000 and 2010. The study was approved by the hospital’s human research committee, and written informed consent for all participants was provided by the participant or an authorized representative.

Figure 1. Segregation of RNF216 and OTUD4 Mutations in the Index Pedigree and Identification of Additional RNF216 Mutations in Unrelated Probands.

The seven-generation pedigree shown in Panel A includes Patients 1, 2, and 3, all of whom presented with ataxia, dementia, and hypogonadotropic hypogonadism and were homozygous for both RNF216 p.R751C and OTUD4 p.G333V. Double lines indicate consanguineous unions. Genotyped, unaffected family members are shown to be either homozygous for the nonmutated alleles (denoted with a + symbol) or heterozygous for one or both changes. The pedigrees shown in Panel B are for the families of additional RNF216 mutation-positive patients (Patients 4 through 8), all of whom presented with ataxia and hypogonadotropic hypogonadism. Squares denote male family members, circles female family members, solid symbols affected family members, slashes deceased family members, diamonds siblings of either sex, the triangle miscarriages, and Arabic numbers the number of siblings or miscarriages.

GENETIC ANALYSIS

We performed exome sequencing with DNA from Patient 3 in the index family. The data sets used for exome analysis in this study were obtained from dbGaP at www.ncbi.nlm.nih.gov/gap through dbGaP accession number phs000475. v1.p1. Candidate genes were sequenced in family members and in unrelated affected persons. Computer algorithms were used to predict the pathogenicity of variants and to identify interactions between candidate genes and genes known to be associated with ataxia or hypogonadotropic hypogonadism. Allele-specific reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays were performed with RNA from Patients 5, 6, and 7 (see the Methods section in the Supplementary Appendix, available with the full text of this article at NEJM.org).

NEUROPATHOLOGICAL AND ENDOCRINE EVALUATION

The brain of Patient 2 was obtained within 6 hours after death. Immunohistochemical analysis was performed with the use of antibodies against ubiquitin, tau, and α-synuclein. Electron microscopy was performed according to standard procedures. Detailed reproductive endocrine pheno-typing was performed in 5 patients, as described in our previous report¹² and in the Methods section in the Supplementary Appendix.

ZEBRAFISH INVESTIGATIONS

Morpholino oligonucleotides (MO) for the silencing of zebrafish rnf216 and otud4 were injected either alone or with nonmutant or mutant human messenger RNA (mRNA) encoding RNF216, mRNA encoding OTUD4, or both (see the Methods section in the Supplementary Appendix).

RESULTS

GENETIC STUDIES

The consanguineous pedigree of the index family (a self-reported Palestinian family) includes three siblings (Patients 1, 2, and 3) with ataxia and hypogonadotropic hypogonadism. Exome sequencing performed with DNA from Patient 3 identified 13 homozygous variants that were rare and predicted to be deleterious (Table S1 in the Supplementary Appendix), 2 of which were also homozygous in the two other affected siblings: RNF216 (NM_207111.3) c.2251C→T, p.R751C and OTUD4 (NM_001102653.1) c.998G→T, p.G333V; these variants were not identified or were heterozygous in the unaffected family members (Fig. 1). RNF216 encodes an E3 ubiquitin-protein ligase. The R751 residue of RNF216 resides within the second of two domains called “really interesting new gene” (RING) finger domains and is conserved across vertebrates (Fig. S1 in the Supplementary Appendix). The R751C variant is predicted to be deleterious by four prediction programs and is absent in 13,006 control chromosomes from the National Heart, Lung, and Blood Institute’s Exome Sequencing Project (ESP) and in 672 chromosomes from Middle Eastern persons (including 36 chromosomes from Palestinian persons). OTUD4 encodes a deubiquitinase (Fig. S1 in the Supplementary Appendix). The G333V variant is predicted to be deleterious by three of four prediction programs and is found in 2 of the 13,006 chromosomes from the ESP and in none of the 672 chromosomes from Middle Eastern persons.

Both OTUD4 and RNF216 were sequenced in nine affected persons from seven unrelated families. No rare variants were identified in OTUD4, but mutations in RNF216 were identified in four probands (Fig. 1, and Fig. S1 in the Supplementary Appendix). Patient 4 had compound heterozygous frameshift and nonsense mutations ([c.615_616delGA; p.E205DfsX15] and [c.1791T→A; p.C597X]). Three additional heterozygous mutations were identified: c.414delG, p.G138GfsX74 in Patients 5 and 6 (siblings); c.721C→T, p.Q241X in Patient 7; and c.2149C→T, p.R717C in Patient 8; the missense mutation in Patient 8 was predicted to be deleterious by four prediction programs.

None of these RNF216 variants were present in the ESP, which did identify five other variants considered to be overtly deleterious and nine considered likely to be deleterious. Given this background level of genetic variation, the presence of deleterious mutations in five of eight probands in this study exceeded what would be expected by chance (P<1×10⁻¹³ by Fisher’s exact test). Grail,¹⁴ DAPPLE,¹⁵ Endeavour,¹⁶ InWeb scored network,¹⁷ and CNVconnect¹⁸ were used to identify potential connections among RNF216, OTUD4, and genes known to be associated with ataxia or hypogonadotropic hypogonadism; no such connections were found. Allele-specific RT-PCR assays failed to identify occult mutations affecting transcription or mRNA stability in the nonmutant alleles of Patients 5, 6, and 7 (Fig. S2 in the Supplementary Appendix).

FUNCTIONAL TESTING

Zebrafish were used to interrogate the functional consequences and suggestive epistatic interactions of the RNF216 and OTUD4 mutations.^19,20 The injection of a MO that disrupted the splicing of zebrafish rnf216 (Fig. S3 in the Supplementary Appendix) caused a reduction in the size of the eye cup and optic tecta and disorganization of the cerebellum, as well as a slight reduction in overall head size (Fig. 2 and Fig. 3, and Fig. S4 in the Supplementary Appendix). The tectal phenotype was rescued by the coinjection of human RNF216 mRNA, but the injection of human RNF216 mRNA encoding R751C failed to rescue the phenotype (Fig. 2), suggesting that the mutation affects the function of the encoded protein. The injection of a splice-blocking MO against zebrafish otud4 (Fig. S3 in the Supplementary Appendix) also induced a reduction in size of the optic tecta and cerebellum (Fig. 3).

Figure 2. Functional Studies of rnf216 in Zebrafish.

Panels A through D show dorsal views of control zebrafish embryos (Panel A) and embryos injected with rnf216 morpholino oligonucleotides (MO) (Panel B), rnf216 MO plus nonmutant human RNF216 (Panel C), and rnf216 MO plus mutant human RNF216 (with RNF216 carrying the p.R751C mutation identified in the index pedigree) (Panel D) at 3 days after fertilization (staining with an antibody against α acetylated tubulin). The circles outline the area of the optic tectum, the structure on which all measurements were based. The bar graph in Panel E shows the relative size of the optic tectum in control embryos and the embryos injected with rnf216 MO, rnf216 MO plus nonmutant human RNF216, and rnf216 MO plus mutant human RNF216. P values are based on two-tailed t-tests. I bars indicate standard errors. AU denotes arbitrary units.

Figure 3. Epistatic Effects of the OTUD4 p.G333V Allele.

Panels A through F show dorsal views of control zebrafish embryos (Panel A) and embryos injected with rnf216 MO (morpholino oligonucleotides) (Panel B), otud4 MO (Panel C), double MO (DMO, rnf216 MO plus otud4 MO) (Panel D), double MO plus nonmutant human OTUD4 (Panel E), and double (DMO) plus mutant human OTUD4 (OTUD4 carrying the p.G333V mutation identified in the index pedigree) (Panel F) at 3 days after fertilization (anti-α acetylated tubulin stain). The asterisks indicate the optic tecta that were measured to assess the differences between the conditions being evaluated. The bar graph in Panel G shows the mean relative size of the optic tecta in control embryos and the five groups of injected embryos. I bars indicate standard errors. P values are based on two-tailed t-tests. Panels H, I, and J show dorsal views of control embryos (Panel H) and embryos injected with DMO (Panel I) and DMO plus nonmutant human OTUD4 (Panel J) at 3 days after fertilization (anti-α acetylated tubulin stain). The rectangles outline the cerebellar area; maximum disorganization is observed in embryos injected only with DMO (Panel I). The bar graph in Panel K shows the percentage of embryos with cerebellar defects under the conditions being evaluated (as shown in Panels A through F and Panels H, I, and J).

Coinjection of both rnf216 and otud4 MOs induced a significant reduction in the size of the optic tecta as compared with the injection of the rnf216 MO alone (P<0.001) (Fig. 3). Double-MO injection also caused marked disorganization of the cerebellum in more than 60% of embryos (Fig. 3) and the development of a severe cerebellar phenotype with complete loss of structural integrity in approximately 30% of embryos, as compared with only 5 to 10% of embryos injected with either the rnf216 or the otud4 MO alone (data not shown). Furthermore, double-MO injection resulted in marked microphthalmia as compared with modest microphthalmia when either MO injection was used alone (Fig. S4 in the Supplementary Appendix). These phenotypes were specific, since coinjection of nonmutant human RNF216 or OTUD4 mRNA rescued all phenotypes (Fig. 3, and Fig. S4 in the Supplementary Appendix). RNF216 mRNA encoding R751C and OTUD4 mRNA encoding G333V were less effective in rescuing the phenotypes induced by double-MO injection (Fig. 3, and Fig. S4 in the Supplementary Appendix), suggesting not only that these mutant alleles encode functionally deficient proteins but also that epistatic interactions between these mutations contribute to the disease phenotype in the index pedigree.

CLINICAL CHARACTERISTICS OF THE STUDY PATIENTS

Patients 1 through 8, who carried variants in RNF216, had similar clinical histories (Table 1). They presented in adolescence or early adulthood with hypogonadotropic hypogonadism but no other pituitary abnormalities. Dysarthria was the initial neurologic symptom in some patients, but ataxia developed in all patients, leading to wheelchair dependency and to bed confinement for some patients. Dementia was also prominent, with personality changes and memory loss occurring at the onset of the disease and mutism and uncoordinated, purposeless movements during the end stages. Nystagmus was absent. The presentation of Patients 9 through 12, who did not have variants in RNF216, was quite different from that of Patients 1 through 8 (Table 1). Extensive evaluation did not reveal any known causes of ataxia in any of the patients; mitochondrial abnormalities were identified in Patients 7 and 8 (Table S2 in the Supplementary Appendix).

Table 1.

Clinical Phenc itypes and RNF216 and OTUD4 Genotypes.^*

Patient and Race or Ethnic Group	Sex	Clinical Features	Imaging Findings	RNF216 genotype	OTUD4 Genotype
Family 1, Palestinian
Patient 1	Male	No spontaneous puberty; at 22 yr, dysarthria, followed   by progressive ataxia and dementia; at 43 yr, death   (aspiration pneumonia)	At 30 yr, CT revealed prominent cerebellar and mild   cortical atrophy, with hypodensities in cerebral   white matter	R751C+R751C	G333V + G333V
Patient 2	Female	At 16 yr, menarche, followed by secondary amenorrhea;   at 20 yr, personality change; at 30 yr, dysarthria,   followed by progressive ataxia and dementia;   at 41 yr, death (aspiration pneumonia)	At 30 yr, CT revealed prominent cerebellar and mild   cortical atrophy, with hypodensities in cerebral   white matter	R751C+ R751C	G333V + G333V
Patient 3	Male	Normal puberty; at 20 yr, erectile dysfunction; at 29 yr,   dysarthria, followed by progressive ataxia and de-   mentia; at 47 yr, death (possibly from pulmonary   embolism)	At 35 yr, MRI revealed diffuse parenchymal volume loss   in cerebellum and cerebral cortex, with multiple   punctate and confluent areas of hyperintensity   on T2-weighted and FLAIR imaging	R751C+R751C	G333V + G333V
Family 2, white
Patient 4	Male	No spontaneous puberty; at 22 yr, dysarthria, ataxia,   and dementia; at 30 yr, prominent chorea; at 36 yr,   death	At 23 yr, MRI revealed cerebellar atrophy and widespread   foci of hyperintensity in cerebral white matter and   thalami	C597X + E205DfsX15	Nonmutant + Nonmutant
Family 3, white
Patient 5	Male	Normal puberty; at 36 yr, hypogonadotropism and chorea   followed by progressive ataxia and dementia	At 42 yr, MRI revealed global atrophy, with diffusely   scattered periventricular foci of hyperintensity on   T2-weighted and FLAIR imaging	G138GfsX74 + Nonmutant	Nonmutant + Nonmutant
Patient 6	Female	Normal puberty; at 27 yr, oligomenorrhea, followed by   amenorrhea; memory problems; at 31 yr, chorea,   followed by progressive ataxia and dementia	At 31 yr, MRI revealed mild-to-moderate cerebellar   atrophy and mild prominence of ventricles and   sulci, with multiple small foci of hyperintensity   on T2-weighted imaging	G138GfsX74 + Nonmutant	Nonmutant+ Nonmutant
Family 4, white
Patient 7	Female	Primary amenorrhea; at 27 yr, ataxia and dysarthria,   followed by progressive ataxia and dementia	At 18 yr, MRI revealed a normal cerebellum and multiple   foci of hyperintensity in subcortical white matter on   T2-weighted imaging; at 35 yr, MRI revealed marked   cerebellar atrophy, with an increased number of   foci of T2-weighted hyperintensity	Q241X + Nonmutant	Nonmutant+ Nonmutant
Family 5, white
Patient 8	Male	No spontaneous puberty; at 19–21 yr, partial response   to treatment with exogenous pulsatile GnRH; at   21 yr, slurred speech and imbalance, followed by   progressive ataxia, mood changes, and memory   impairment	At 17 yr, MRI revealed slight prominence of fissures in   cerebellum; at 23 yr, M Rl revealed severe cerebellar and   mild cerebral atrophy, multiple foci of T2-weighted   and FLAIR hyperintensity	R717C + Nonmutant	Nonmutant+ Nonmutant
Family 6, white
Patient 9	Male	No spontaneous puberty; at 5 yr, ataxia, nystagmus;   at 34 yr, still able to walk		Nonmutant + Nonmutant	Nonmutant+ Nonmutant
Patient 10	Male	No spontaneous puberty; at 5 yr, ataxia, nystagmus;   at 32 yr, still able to walk		Nonmutant + Nonmutant	Nonmutant+ Nonmutant
Family 7, white
Patient 11	Male	No spontaneous puberty; at 4 yr, ataxia; at 28 yr, ataxia   stable	At 4 yr, CT revealed normal cerebellum; at 17 yr, MRI   revealed cerebellopontine atrophy, global white-   matter abnormality in basal ganglia and cortex	Nonmutant+ Nonmutant	Nonmutant+ Nonmutant
Family 8, Asian
Patient 12	Female	Normal puberty; at 17 yr, behavior problems, followed by   tremor, dysarthria, and ataxia; at 19 yr, internuclear   ophthalmoplegia; at 24 yr, hypogonadotropism	At 21 yr, MRI revealed mild atrophy, T2-weighted and   FLAIR imaging revealed hyperintensity in brain   stem, thalami, internal capsules, insulae, basal   ganglia, and periventricular regions	Nonmutant+ Nonmutant	Nonmutant + Nonmutant

^*CT denotes computed tomography, FLAIR fluid-attenuated inversion recovery, GnRH gonadotropin-releasing hormone, and MRI magnetic resonance imaging.

Neuroimaging performed in Patients 1 through 8 revealed striking similarities, with cerebellar and cortical atrophy but no abnormalities of the pituitary gland. The subcortical white matter contained patchy areas of hyperintensity on T₂-weighted imaging and fluid-attenuated inversion recovery (FLAIR) imaging (Table 1 and Fig. 4). In Patient 7, these areas of hyperintensity were present approximately 9 years before the onset of neurologic symptoms; the cerebellum appeared normal at that earlier point in time.

Figure 4. Neuroradiologic and Neuropathological Findings.

Panel A shows a sagittal T₂-weighted magnetic resonance imaging scan of the brain in Patient 3. Diffuse cerebellar atrophy (arrow) and cortical atrophy can be seen. Panel B shows a transverse image obtained with fluid-attenuated inversion recovery imaging, revealing multiple distinct and confluent foci of hyperintensity in the white matter. In Panel C, immunohistochemical analysis of a hippocampal brain section from Patient 2 shows a neuronal intranuclear inclusion with immunoreactivity (brown) to an antibody against ubiquitin, counterstained with hematoxylin and eosin. An electron micrograph of the hippocampal neurons, in Panel D, also shows an intranuclear inclusion, which consists of aggregates of granular material and fine filaments, 10 to 15 nm in diameter (arrow), that are for the most part randomly oriented. The scale bar corresponds to 1 µm.

NEUROPATHOLOGICAL STUDIES

The formalin-fixed brain of Patient 2 weighed 940 g (normal weight, 1300 g). The cerebellum and inferior olives were atrophic. Histopathological analysis revealed gliosis and virtually complete loss of inferior olivary neurons, cerebellar Purkinje’s cells, and neurons in hippocampal regions CA3 and CA4, whereas neurons were well preserved in regions CA1 and CA2. Ubiquitin-immunoreactive nuclear inclusions were present in 1 to 5% of the pyramidal neurons in hippocampal regions CA1 and CA2 (Fig. 4) and were also found in granule-cell neurons in the dentate gyrus; these inclusions were not immunoreactive to antibodies against tau or α-synuclein (not shown). On electron microscopy, the intranuclear inclusions appeared as aggregates of fine filaments and granular material (Fig. 4).

REPRODUCTIVE ENDOCRINE STUDIES

When Patient 6 reached 32 years of age, 1 year after the development of neurologic symptoms, low-amplitude pulses of luteinizing hormone were detected, indicating that GnRH secretion, although present, was diminished (Fig. 5). The administration of pulsatile GnRH for 7 days induced robust increases in levels of gonadotropins and estradiol (Fig. 5) as well as the growth of a dominant ovarian follicle, observed on ultraso-nography (not shown). Although the secretion of luteinizing hormone increased in response to the administration of GnRH, the typical peaked pattern of luteinizing hormone pulses²¹ was not seen, which suggested a degree of pituitary dysfunction. Indeed, the patient’s pituitary responsiveness waned over time, with a diminished response to GnRH on day 1 of treatment 15 months after the initial endocrine study (Fig. 5).

Figure 5. Endocrine Phenotypes.

In Panels A through D, the graphs at the left show the endogenous secretion of luteinizing hormone over a period of up to 12 hours. Patient 6 was studied on two occasions, 15 months apart (Panels A and B). Arrowheads indicate pulses of luteinizing hormone secretion, and boxes duration of sleep; the shading indicates the reference range for healthy men and women. Concentrations of estradiol (E2) and testosterone (T), measured from pooled samples obtained during the study, are indicated. In Panels A, B, and D, the graphs at the right show the response to exogenous pulsatile gonadotropin-releasing hormone (GnRH) over the course of up to 7 days. The dose of GnRH was 75 ng per kilogram of body weight, with the exception of the first dose of GnRH on day 1 for Patient 6 (Panel A), which was 165 ng per kilogram. (Note the difference in the y axis scales in Panels A and B.) In Panel C, the graph at the right shows the secretion of lutein-izing hormone in response to varying doses of GnRH (black circles and regression line). The data for the patient fall to the right of the 95% confidence interval (dashed red lines) for the mean amplitude of the response to a range of GnRH doses in 6 other men with idio-pathic hypogonadotropic hypogonadism (solid red line).

In Patient 8, in whom endocrine function was initially assessed before the onset of neurologic symptoms, there was an absence of endogenous pulsatile luteinizing hormone secretion (Fig. 5). After escalating doses of exogenous GnRH (from 25 ng per kilogram of body weight to 600 ng per kilogram every 2 hours), the patient’s testicular volume increased from 2 ml to 8 ml but did not increase further, despite these very high doses. Although his pituitary response to exogenous treatment with GnRH was impaired (Fig. 5), direct gonadal stimulation with human chorionic gonadotropin normalized the testosterone level, at 459 ng per deciliter (15.9 nmol per liter).

Pituitary responsiveness to GnRH was lost in patients late in the course of their disease. In Patients 1 and 2, who were bedridden when pituitary responsiveness was assessed, there was no detectable luteinizing hormone secretion and no measurable change in the gonadotropin level in response to the administration of pulsatile exogenous GnRH (Fig. 5).¹²

DISCUSSION

The underpinnings for the association of ataxia with hypogonadotropic hypogonadism have eluded investigators for more than a century. This report shows that ataxia with hypogonadotropic hypogonadism can be caused by mutations in RNF216 either singly or in combination with mutations in OTUD4. Both RNF216 and OTUD4 encode proteins that regulate ubiquitination, indicating that abnormalities in this fundamental cellular process can have pathologic effects on the cerebellum and hippocampus, the cerebral white matter, and the hypothalamic and pituitary components of the reproductive endocrine cascade.

The compound heterozygous termination mutations in RNF216 in Patient 4 firmly implicate RNF216 as a causative gene for this syndrome. Heterozygous RNF216 mutations were found in Patients 5 through 8 but did not cause disease in their parents. Oligogenicity offers a parsimonious explanation for these observed patterns. Oligogenic inheritance has been described in the Bardet–Biedl and Bartter syndromes, Hirsch-sprung’s disease, and isolated hypogonadotropic hypogonadism.^19,22–27 In such an oligogenic model, RNF216 mutations can act with mutations at other genetic loci to cause disease.

Indeed, the phenotype in the index pedigree appears to have been caused by the interaction of hypomorphic mutations in RNF216 and OTUD4. Inhibition of either RNF216 or OTUD4 in zebrafish resulted in cerebellar hypoplasia, microphthalmia, and small optic tecta. The concordance of these phenotypes suggests that RNF216 and OTUD4 operate in the same pathway. This possibility is bolstered by the observation of epistatic interactions between RNF216 and OTUD4, with simultaneous knockdown of both genes resulting in more severe phenotypes. Taken together, these findings support a digenic model in which the OTUD4 mutation, in conjunction with the RNF216 mutation, played an essential role in causing disease in the index pedigree. By extension, in Patients 5 through 8, it is possible that mutations in other, currently unidentified loci may have acted in conjunction with the heterozygous mutations in RNF216 to cause disease. Oligogenicity is likely to be increasingly recognized as methods for detecting this genetic architecture, such as exome sequencing, are more widely adopted.

RNF216 encodes an E3 ubiquitin ligase that attaches ubiquitin to protein substrates, marking them for proteasome-mediated degradation. Known targets of RNF216 include upstream activators of nuclear factor κB signaling, which regulates diverse cellular processes.^28–31 RNF216 is structurally similar to parkin, an E3 ubiquitin ligase that is mutated in a recessive form of Parkinson’s disease.^32–34 The finding of neuronal intranuclear inclusions in Patient 2 may indicate that RNF216-associated neurodegeneration has similarities not only with Parkinson’s disease but also with other neurodegenerative disorders in which protein aggregates are found, such as Huntington’s disease and Alzheimer’s disease.³⁵

OTUD4 encodes a deubiquitinating enzyme. Deubiquitinases allow target proteins and ubiquitin itself to be recycled and often function in partnership with specific E3 ligases. For example, the deubiquitinase ataxin-3 counteracts the ability of parkin to ubiquitinate itself.³⁶ On the basis of this and other examples,³⁷ OTUD4 and RNF216 may be similarly linked in a coregulatory partnership.

The progressive and debilitating dementia observed in the patients with RNF216 mutations (Patients 1 through 8) distinguishes them from the other patients with ataxia and hypogonadotropic hypogonadism. Furthermore, we observed changes in cerebral white matter in all the patients with RNF216-associated neurodegeneration, suggesting that such changes may constitute a consistent feature of this syndrome. None of these patients had oculomotor abnormalities such as the nystagmus and ophthalmoplegia seen in Patients 9, 10, and 12, who did not have RNF216 mutations.

The patients with RNF216-associated neuro-degeneration had dysfunction at multiple levels of the reproductive endocrine axis. In Patients 6 and 8, reproductive function was restored with extended GnRH treatment, which suggests that hypothalamic GnRH deficiency was the primary cause of their reproductive endocrine dysfunction. However, these two patients also appeared to have an element of pituitary dysfunction, given the diminishing responses to GnRH over time in Patient 6 and the observation of a right-shifted dose–response curve in Patient 8. In Patients 1 and 2, who were evaluated late in the course of their disease, the complete absence of response after 7 days of treatment with GnRH may represent progression of this pituitary dysfunction. The basis for the selective vulnerability of particular neuronal and pituitary cell types is currently unexplained.

In conclusion, we have identified loss-of-function mutations in RNF216 that cause a syndrome of ataxia, dementia, and hypogonadotropic hypogonadism. Genetic and in vivo evidence suggests that mutations affecting RNF216, an E3 ubiquitin ligase, and OTUD4, a deubiquitinase, can synergize to cause this syndrome, reinforcing the notion that the mutational load within biologic pathways can drive disease manifestation.²⁰ Taken together, these data highlight a hitherto unknown role of the ubiquitination system in disorders of combined neurodegeneration and reproductive dysfunction. More broadly, our findings show the value of combining individual whole-exome sequencing with in vivo functional studies to identify disease-causing gene mutations and epistatic interactions.