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.).

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.

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).

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.

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).