Genetic and phenotypic characterization of NKX6-2-related spastic ataxia and hypomyelination

Abstract

Background and purpose: Hypomyelinating leukodystrophies are a heterogeneous group of genetic disorders with a wide spectrum of phenotypes and a high rate of genetically unsolved cases. Bi-allelic mutations in NKX6-2 were recently linked to spastic ataxia 8 with hypomyelinating leukodystrophy.

Methods: Using a combination of homozygosity mapping, exome sequencing, and detailed clinical and neuroimaging assessment a series of new NKX6-2 mutations in a multicentre setting is described. Then, all reported NKX6-2 mutations and those identified in this study were combined and an in-depth analysis of NKX6-2-related disease spectrum was provided.

Results: Eleven new cases from eight families of different ethnic backgrounds carrying compound heterozygous and homozygous pathogenic variants in NKX6-2 were identified, evidencing a high NKX6-2 mutation burden in the hypomyelinating leukodystrophy disease spectrum. Our data reveal a phenotype spectrum with neonatal onset, global psychomotor delay and worse prognosis at the severe end and a childhood onset with mainly motor phenotype at the milder end. The phenotypic and neuroimaging expression in NKX6-2 is described and it is shown that phenotypes with epilepsy in the absence of overt hypomyelination and diffuse hypomyelination without seizures can occur.

Conclusions: NKX6-2 mutations should be considered in patients with autosomal recessive, very early onset of nystagmus, cerebellar ataxia with hypotonia that rapidly progresses to spasticity, particularly when associated with neuroimaging signs of hypomyelination. Therefore, it is recommended that NXK6-2 should be included in hypomyelinating leukodystrophy and spastic ataxia diagnostic panels.

Keywords: NKX6-2; SPAX8; hypomyelination; leukodystrophy; spastic ataxia 8.

Figure 1

Family trees in all new families reported in this study. het, heterozygous; hom, homozygous; the individuals tested in this study are indicated with a dot.

Figure 2

Mutation spectrum of NKX6‐2‐related disease. (a) NKX6‐2 gene with all the mutations identified. All known and novel mutations identified in our cohort are labelled with a blue star; all mutations previously reported are labelled with a magenta star and plotted on top of the gene. (b) Sanger sequencing confirmation with segregation analysis for novel NKX6‐2 variants reported in this study. (c) Homozygosity mapping in family IV and V identified a homozygous region on chromosome 10, shared by affected individuals and containing the pathogenic homozygous variant c.196delC in NKX6‐2. (d) Conservation across species of each novel missense mutation reported in this study.

Figure 3

Functional analyses and pathogenicity of variants identified in family 1. (a) Reverse transcription polymerase chain reaction in compound heterozygous missense case F1‐III:1 showed absent or severely reduced NKX6‐2 compared to control; glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control. (b) Reduced NKX6‐2 protein levels confirmed by western blot in individual F1‐III:1. Total protein lysate extracted from human fibroblasts assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by western blotting using anti‐NKX6‐2 antibody (left panel). (c) Densitometry analysis shows significant reduction in NKX6‐2 protein levels in fibroblasts harbouring the NKX6‐2 missense mutation compared to control cells. ***P < 0.01, replicate values, mean and SD are shown; one‐way anova with Bonferroni post hoc test.

Figure 4

Genetype–phenotype correlation and neuroimaging spectrum of NKX6‐2 mutations. (a) The neonatal‐onset group has a statistically significant higher frequency of global psychomotor developmental delay (red) compared with the other two groups (onset from 2 months to 1 year and onset after 1 year). Childhood onset is associated with predominantly motor delay (blue). P = 0.05, r ² = 0.9. (b) Clinical features associated with NKX6‐2 mutations (the horizontal axis) with the number of cases on the vertical axis (total n = 33). hor, horizontal gaze‐evoked nystagmus. (c) Fluid‐attenuated inversion recovery and T2‐weighted MRI acquisitions from case F1‐III:1 exhibiting T2 hyperintense signal change in periventricular WM surrounding the frontal horn of the right lateral ventricle, and frontal and temporal opercular and subinsular WM T2‐weighted hyperintense signal change associated with a degree of cortical volume loss. Note the normal signal intensity of the globi pallidi, thalami and external capsules, mesencephalon and pons. There is disproportionate cerebellar volume loss with mild T2‐weighted hyperintense signal change in the peri‐dentate WM. (d) Longitudinal MRI in case F7‐II:3 at ages of 4 years (D1, D2) and 8½ years (D3, D4) (D1, D3, mid‐sagittal T1‐weighted; D2, D4, coronal T2‐weighted) showing progressive thinning of the corpus callosum and cerebellar atrophy associated with WM abnormality sparing the U fibres (2, 4). There is progressive enlargement of the cortical sulci and extra axial cerebrospinal fluid spaces indicating underlying global brain atrophy.