PNPLA6 mutations cause Boucher-Neuhauser and Gordon Holmes syndromes as part of a broad neurodegenerative spectrum

Abstract

Boucher-Neuhäuser and Gordon Holmes syndromes are clinical syndromes defined by early-onset ataxia and hypogonadism plus chorioretinal dystrophy (Boucher-Neuhäuser syndrome) or brisk reflexes (Gordon Holmes syndrome). Here we uncover the genetic basis of these two syndromes, demonstrating that both clinically distinct entities are allelic for recessive mutations in the gene PNPLA6. In five of seven Boucher-Neuhäuser syndrome/Gordon Holmes syndrome families, we identified nine rare conserved and damaging mutations by applying whole exome sequencing. Further, by dissecting the complex clinical presentation of Boucher-Neuhäuser syndrome and Gordon Holmes syndrome into its neurological system components, we set out to analyse an additional 538 exomes from families with ataxia (with and without hypogonadism), pure and complex hereditary spastic paraplegia, and Charcot-Marie-Tooth disease type 2. We identified four additional PNPLA6 mutations in spastic ataxia and hereditary spastic paraplegia families, revealing that Boucher-Neuhäuser and Gordon Holmes syndromes in fact represent phenotypic clusters on a spectrum of neurodegenerative diseases caused by mutations in PNPLA6. Structural analysis indicates that the majority of mutations falls in the C-terminal phospholipid esterase domain and likely inhibits the catalytic activity of PNPLA6, which provides the precursor for biosynthesis of the neurotransmitter acetylcholine. Our findings show that PNPLA6 influences a manifold of neuronal systems, from the retina to the cerebellum, upper and lower motor neurons and the neuroendocrine system, with damage of this protein causing an extraordinarily broad continuous spectrum of associated neurodegenerative disease.

Keywords: ataxia; early onset ataxia; genetics; hereditary spastic paraplegia; hypogonadism; recessive ataxia; retinal degeneration; spastic ataxia; spasticity.

Figure 1

Representation of identified PNPLA6 mutations and structural analysis of their effect on the PNPLA6 protein. (A) Schematic of the exon-intron arrangement of PNPLA6 (NCBI reference NM_001166111.1), with positions of mutations identified in five families. Exons are indicated as black boxes. CNB1/2 and the phospholipid esterase functional domains are indicated by purple and orange boxes, respectively. The mutations are indicated and colour-coded by phenotype observed. (B) The inter-species conservation of the amino acids affected by missense mutations identified in this study. (C) Structural model of EST domain (grey) of human PNPLA6. The side chain moieties of active site residues (Ser1014/Asp1144) and those identified here to be prone to mutations (Ser1045/Thr1058/Phe1066/Val1100/Val110/Pro1122) are coloured red and blue, respectively. The funnel-shaped catalytic centre of the EST domain—where the active site residues Ser1014/Asp1144 are located deep at the base of the funnel whereas the rim of the funnel represents the entry route for the phosphatidylcholine (PDC)_substrate (green arrow)—is represented by yellow dashed lines. Note that the structural model of EST domain was built using the crystal structure of the homologous EST catalytic domain of the plant patatin PAT17 (PDB# 1OXW) (Rydel et al., 2003) in MODELLER (Marti-Renom et al., 2000). A total of 100 atomic models were calculated and the structure with the lowest energy, as judged by the MODELLER Objective Function, was selected for further analysis. The structural model was rendered using RIBBONS (Carson, 1991) (for a larger version and an additional rotation perspective, see Supplementary Fig. 3).

Figure 2

The continuous spectrum of PNPLA6-associated disease (A) and the classical clinical trias of the Boucher-Neuhäuser syndrome (B–D). (A) The clinical spectrum of PNPLA6 mutations unfolds along four different neurological key features: ataxia, chorioretinal dystrophy, hypogonadotropic hypogonadism and motor neuron disease (upper motor neuron disease with or without additional lower motor neuropathy). Accordingly, Boucher-Neuhäuser syndrome (BNS) and Gordon Holmes syndrome (GHS) are not distinct entities, but clusters on a continuous spectrum of PNPLA6-associated disease, extending from Boucher-Neuhäuser syndrome via Gordon Holmes syndrome to spastic ataxia (sATX) and pure hereditary spastic paraplegia (HSP). The phenotype of complicated hereditary spastic paraplegia, which has been considered the most prominent phenotype of PNPLA6 so far (Rainier et al., 2008), is only the ‘tip of the iceberg’ of this broad disease spectrum. (B) Photograph of the full body of a male patient with Boucher-Neuhäuser syndrome (Subject IHG 25357) at age 26 years illustrating incomplete secondary sex characteristics with lack of body hair and gynecomastia. (C) Exemplary fundus photography of the Boucher-Neuhäuser syndrome Patient ARCA_05 showing chorioretinal degeneration, characterized by diffuse atrophy of choroidal vessels and the retinal pigment epithelium with pigment clumps. The optic nerve shows no signs of atrophy. (D) Sagittal T₂-weighted MRI of this subject with Boucher-Neuhäuser syndrome shows marked cerebellar atrophy.